Regulated angiogenesis genes and polypeptides

ABSTRACT

The present invention relates to all facets of novel polynucleotides, the polypeptides they encode, antibodies and specific binding partners thereto, and their applications to research, diagnosis, drug discovery, therapy, clinical medicine, forensic science and medicine, etc. The polynucleotides are modulated during angiogeneis and are therefore useful in variety of ways, including, but not limited to, as molecular markers, as drug targets, and for detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating, determining predisposition to, etc., diseases and conditions, determining predisposition to, etc., diseases and conditions, such as abnormal, insufficient, excessive, etc., angiogenesis, inflammatory diseases, rheumatoid arthritis, osteoarthritis, asthma, pulmonary fibrosis, age-related macular degeneration (ARMD), diabetic retinopathy, macular degeneration, and retinopathy of prematurity (ROP), endometriosis, cancer, Coats&#39; disease, peripheral retinal neovascularization, neovascular glaucoma, psoriasis, retrolental fibroplasias, angiofibroma, inflammation, etc.

DESCRIPTION OF THE DRAWINGS

FIGS. 1-19 show amino acid sequence alignments between polypeptides ofthe present invention, and polypeptides listed in public databases. SEQID NOS for the polypeptides of the present invention are listed in Table3. Others are as follows: XM_(—)087061 (SEQ ID NO 59), X68560 (SEQ ID NO60), XM_(—)045848 (SEQ ID NO 61), XM_(—)055371 (SEQ ID NO 62),XM_(—)076374 (SEQ ID NO 63), AL133047 (SEQ ID NO 64), XM_(—)086643 (SEQID NO 65), AK027731 (SEQ ID NO 66), AK000913 (SEQ ID NO 67),NM_(—)004713 (SEQ ID NO 68), XM_(—)053487 (SEQ ID NO 69), NM 000366 (SEQID NO 70), AL133087 (SEQ ID NO 71), AF326966 (SEQ ID NO 72),NM_(—)014882 (SEQ ID NO 73), XM_(—)015539 (SEQ ID NO 74), XM_(—)087631(SEQ ID NO 75), XM_(—)048092 (SEQ ID NO 76), NM 052877 (SEQ ID NO 77),XM_(—)001738 (SEQ ID NO 78); AAF19255 (SEQ ID NO 79); AAC97961 (SEQ IDNO 80).

DESCRIPTION OF THE INVENTION

The present invention relates to all facets of novel polynucleotides,the polypeptides they encode, antibodies and specific binding partnersthereto, and their applications to research, diagnosis, drug discovery,therapy, clinical medicine, forensic science and medicine, etc. Thepolynucleotides are expressed in angiogenesis and are therefore usefulin variety of ways, including, but not limited to, as molecular markersfor blood vessels and blood vessel formation, as drug targets, and fordetecting, diagnosing, staging, monitoring, prognosticating, preventing,treating, and/or determining predisposition to diseases and conditionsof the vascular system. The identification of specific genes, and groupsof genes, expressed in pathways physiologically relevant to angiogenesispermits the definition of functional and disease pathways, and thedelineation of targets in these pathways which are useful in diagnostic,therapeutic, and clinical applications. The present invention alsorelates to methods of using the polynucleotides and related products(proteins, antibodies, etc.) in business and computer-related methods,e.g., advertising, displaying, offering, selling, etc., such productsfor sale, commercial use, licensing, etc.

Angiogenesis, the process of blood vessel formation, is a key event inmany physiological processes that underlie normal and diseased tissuefunction. During ontogeny, angiogenesis is necessary to establish to thenetwork of blood vessels required for normal cell, tissue and organdevelopment and maintenance. In the adult organism, the production ofnew blood vessels is needed for organ homeostasis, e.g., in the cyclingof the female endometrium, for blood vessel maturation during woundhealing, and other processes involved in the maintenance of organismintegrity. It also is important in regenerative medicine, including,e.g., in promoting tissue repair, tissue engineering, and the growth ofnew tissues, inside and outside the body.

Not all angiogenesis is beneficial. Inappropriate and ectopic expressionof angiogenesis can be deleterious to an organism. A number ofpathological conditions are associated with the growth of extraneousblood vessels. These include, e.g., diabetic retinopathy, neovascularglaucoma, psoriasis, retrolental fibroplasias, angiofibroma,inflammation, etc. In addition, the increased blood supply associatedwith cancerous and neoplastic tissue, encourages growth, leading torapid tumor enlargement and metastasis.

Because of the importance of angiogenesis in many physiologicalprocesses, its regulation has application in a vast arena oftechnologies and treatments. For instance, induction of neoangiogenesishas been used for the treatment of ischemic myocardial diseases, andother conditions (e.g., ischemic limb, stroke) produced by the lack ofadequate blood supply. See, e.g., Rosengart et al., Circulation,100(5):468-74, 1999. In growth new tissues from progenitor and stemcells, angiogenesis is one of the key processes necessary. Wherevascularization is undesirable, such as for cancer and the mentionedpathological conditions, inhibition of angiogenesis has been used as atreatment therapy. See, e.g., U.S. Pat. No. 6,024,688 for treatingneoplasms using angiogenesis inhibitors.

A number of different factors have been identified which stimulateangiogenesis, e.g., by activating normally quiescent endothelial cells,by acting as a chemoattractant to developing capillaries, by stimulatinggene expression, etc. These factors include, e.g. fibroblast growthfactors, such as FGF-1 and FGF-2, vascular endothelial growth factor(VEGF), platelet-derived endothelial cell growth factor (PD-ECGF), etc.Inhibition of angiogenesis has been achieved using drugs, such asTNP-470, monoclonal antibodies, antisense nucleic acids and proteins,such as angiostatin and endostatin. See, e.g., Battegay, J. Mol. Med.,73, 333-346 (1995); Hanahan et al., Cell, 86, 353-364 (1996); Folkman,N. Engl. J. Med., 333, 1757-1763 (1995).

Activity of a polynucleotide or gene in modulating or regulatingangiogenesis can be determined according to any effective in vivo or invitro methods. One useful model to study angiogenesis is based on theobservation that, when a reconstituted basement membrane matrix, such asMatrigel®, supplemented with growth factor (e.g., FGF-1), is injectedsubcutaneously into a host animal, endothelial cells are recruited intothe matrix, forming new blood vessels over a period of several days.See, e.g., Passaniti et al., Lab. Invest., 67:519-528, 1992. By samplingthe extract at different times, angiogenesis can be temporallydissected, permitting the identification of genes involved in all stagesof angiogenesis, including, e.g., migration of endothelial cells intothe matrix, commitment of endothelial cells to angiogenesis pathway,cell elongation and formation of sac-like spaces, and establishment offunctional capillaries comprising connected, and linear structurescontaining red blood cells. To stabilize the growth factor and/or slowits release from the matrix, the growth factor can be bound to heparinor another stabilizing agent. The matrix can also be periodicallyre-infused with growth factor to enhance and extend the angiogenicprocess.

Other useful systems for studying angiogenesis, include, e.g.,neovascularization of tumor explants (e.g., U.S. Pat. Nos. 5,192,744;6,024,688), chicken chorioallantoic membrane (CAM) assay (e.g., Taylorand Folkman, Nature, 297:307-312, 1982; Eliceiri et al., J. Cell Biol.,140, 1255-1263, 1998), bovine capillary endothelial (BCE) cell assay(e.g., U.S. Pat. No. 6,024,688; Polyerini, P. J. et al., MethodsEnzymol., 198: 440-450, 1991), migration assays, HUVEC (human umbilicalcord vascular endothelial cell) growth inhibition assay (e.g., U.S. Pat.No. 6,060,449).

The present invention relates to polynucleotides, and the polypeptidesthey encode, which are related to angiogenesis and the vascular system.These polynucleotides were identified using a model system forangiogenesis. In this system, a Matrigel™ plug implant comprising FGF-1is implanted subcutaneously into a host mouse. The initial bolus of FGFattracts endothelial cells into the implant, but does not result in newblood vessel formation. After about 10-15 days, the implant isre-infused with FGF-1. The FGF-1 stimulates the endothelial cellsalready present in the implant, initiating the process of angiogenesis.Tissue samples, removed at different intervals, can be analyzed todetermine their gene expression patterns.

In results reported here, samples of the Matrigel™ plug were harvestedimmediately prior to the re-injection with FGF-1, and then 1, 8, and 24hours later. These samples were analyzed for gene expression, anddifferentially-expressed genes were identified by several methods. Atleast eight different expression patterns were observed. These wereclassified according to whether the genes were up-(U) or down-(D)regulated, and whether the expression of the differentially-regulatedgene was transient (T) or sustained (S). The term “transient” indicatesthat the gene expression levels changed temporarily, and then returnedto the basal level. “Sustained” indicates that the expression levelschanged, and then remained relatively stable. The sample removed priorto the FGF-1 re-infusion was used to establish the basal levels of geneexpression, prior to angiogenesis. “L” indicates that expression levelswere low; “H” indicates that expression levels were high. The followingpatterns were observed:

-   -   U1S: Gene up-regulated at 1-hour, and remained up in the 8- and        24-hour assays.    -   U8S: Gene up-regulated at 8-hours, and remained up in 24-hour        assay.    -   U1T: Gene up-regulated at 1-hour, but returned to basal level in        the 8- and 24-hour assays.    -   U8T: Gene up-regulated at 8-hours, but returned to basal level        in the 24-hour assay.    -   D1S: Gene down-regulated at l-hour, and remained down in the 8-        and 24-hour assays.    -   D8S: Gene down-regulated at 8-hours, and remained down in the        24-hour assay.    -   D1T: Gene down-regulated at 1-hour, but returned to basal level        in the 8- and 24-assays.    -   D8T: Gene down-regulated at 8-hours, but returned to basal level        in 24-hour assay.    -   D24: Gene down-regulated in the 24-hour assay.

At the first time point (“0”), endothelial and other cells are presentin the Matrigel™ plug, but angiogenesis has not begun. After 1 hour, theendothelial cells have been stimulated by FGF, and genes involved inangiogenesis have been activated. By 8 hours, the endothelial cells haveorganized into a rudimentary tubes, but are not yet functional. At theend of 24 hours, the tubes have become functional, and are filled withblood cells. SEQ ID NOS 1-58 represent the human homologs ofpolynucleotides identified in this assay system. It should be recognizedthat the specific expression patterns summarized Table 3 reflect thekinetics and particularities of this system.

In accordance with the present invention, genes have been identifiedwhich are differentially expressed in angiogenesis. By the phrase“differential expression,” it is meant that the levels of expression ofa gene, as measured by its transcription or translation product, aredifferent depending upon the time point (see below) in development whenthe cells are assayed. There are no absolute amounts by which the geneexpression levels must vary, as long as the differences are measurable.

The phrase “up-regulated” indicates that an mRNA transcript or othernucleic acid corresponding to a polynucleotide of the present inventionis expressed in larger amounts at a given developmental stage ascompared to another at a given developmental stage as compared toanother. The phrase “down-regulated” indicates that an mRNA transcriptor other nucleic acid corresponding to a polynucleotide of the presentinvention is expressed in lower amounts at a given developmental stageas compared to another.

Not all subjects in an animal population will display the same geneexpression profile, even when they express same or similar phenotypes.For instance, a group of patients may all have a cancer in whichangiogenesis has been initiated, but they may not have 100% identicalpatterns of angiogenic gene expression. There are a number of reasonsfor such differences, including, e.g., variability among patient geneticbackgrounds, differences in their exposure to environmental and otherexogenous factors that influence gene expression, drug histories, cancertype, stage, and grade, allelic variations in the angiogenic genes, etc.For these reasons, there can be circumstances where one gene isinadequate as a general tool to assess and treat angiogenesis. As aresult, it may be desirable to use the genes in combination, rather thanone at a time, to increase the diagnostic and therapeutic efficacy.While one particular gene may not be fully penetrant in all individualsexhibiting angiogenesis, using a set of genes enhances the probabilityof identifying angiogenesis is a broad population sample.

Table 1 is a list of differentially regulated genes in angiogenesis, andtheir corresponding functional and structural polypeptide domains. Table3 summarizes the expression profile of these genes.

The polynucleotide and polypeptide sequences are shown in FIGS. 1-19 andSEQ ID NOS 1-58. Membrane (i.e., cell-surface) proteins coded for byregulated genes (e.g., ANH0668 and ANH0095) are useful targets forantibodies and other binding partners (e.g., ligands, aptamers, smallpeptides, etc.) to selectively target agents to angiogenic tissue forany purpose, included, but not limited to, imaging, therapeutic,diagnostic, drug delivery, gene therapy, etc. For example, bindingpartners, such as antibodies, can be used to block angiogenesis, e.g.,in cancer. Membrane (e.g., when shed into the blood and other fluid) andother differentially expressed proteins can also be used as markers todetermine in a cnacer patient whether angiogenesis in the cancer isprogressing. Especially useful proteins are those which are notexpressed in peripheral blood, such as ANH0144, ANH0459, ANH0687, andANH0316.

Polynucleotides of the present invention have been mapped to specificchromosomal bands. Different human disorders are associated with thesechromosome locations. See, Table 2. The polynucleotides and polypeptidesthey encode can be used as linkage markers, diagnostic targets,therapeutic targets, for any of the mentioned disorders, as well as anydisorders or genes mapping in proximity to them.

The present invention relates to the complete polynucleotide andpolypeptide sequences disclosed herein, as well as fragments thereof.Useful fragments include those which are unique and which do not overlapany known gene, which overlap with a known sequence, which spanalternative splice junctions, which are unique to a public sequence asindicated in the figures, which span an alternative splice junction of apublic sequence, etc. Unique sequences can also be described as beingspecific for a gene because they are characteristic of the gene, but notrelated genes. The unique or specific sequences included polypeptidesequences, coding nucleotide sequences (e.g., as illustrated in thefigures), and non-coding nucleotide sequences.

Below, for illustration, are some examples of polypeptides (included arethe polynucleotides which encode them); however, the present inventionincludes all fragments, especially of the categories mentioned above areexemplified below.

ANH009 (SEQ ID NO 2): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 1-247, 248-378, polypeptidefragments thereof, and polynucleotides encoding said polypeptides;

ANH0024A (SEQ ID NO 4): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 1-93, 94-285, 286-781,285-286, polypeptide fragments thereof, and polynucleotides encodingsaid polypeptides;

ANH0024B (SEQ ID NO 6): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 1-52, 52-53, polypeptidefragments thereof, and polynucleotides encoding said polypeptides;

ANH0024C (SEQ ID NO 8): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 1-496, polypeptide fragmentsthereof, and polynucleotides encoding said polypeptides;

ANH0024D (SEQ ID NO 10): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 1-26, polypeptide fragmentsthereof, and polynucleotides encoding said polypeptides;

ANH0039 (SEQ ID NO 12): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 75-137, polypeptidefragments thereof, and polynucleotides encoding said polypeptides;

ANH0068 (SEQ ID NO 14): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 1-11, 11-12, 12-297,polypeptide fragments thereof, and polynucleotides encoding saidpolypeptides;

ANH0114 (SEQ ID NO 16): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 345, 444-621, 444-472,473-621, polypeptide fragments thereof, and polynucleotides encodingsaid polypeptides;

ANH144A (SEQ ID NO 18): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 1-693, 176, 176-177,610-611, 695-717, 611-693, 938-1070, polypeptide fragments thereof, andpolynucleotides encoding said polypeptides;

ANH144B (SEQ ID NO 20): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 611-718, 802-803, 1001-1023,polypeptide fragments thereof, and polynucleotides encoding saidpolypeptides;

ANH144C (SEQ ID NO 22): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 177-179, polypeptidefragments thereof, and polynucleotides encoding said polypeptides;

ANH0241 (SEQ ID NO 24): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 1-933, 934-1214, polypeptidefragments thereof, and polynucleotides encoding said polypeptides;

ANH0245 (SEQ ID NO 26): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 1-18, 18-359, polypeptidefragments thereof, and polynucleotides encoding said polypeptides;

ANH0296 (SEQ ID NO 28): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 1-518, 518-519, 519-720,721-1082, polypeptide fragments thereof, and polynucleotides encodingsaid polypeptides;

ANH0423 (SEQ ID NO 30): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 596-725, polypeptidefragments thereof, and polynucleotides encoding said polypeptides;

ANH0459B (SEQ ID NO 32): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 41-80, polypeptide fragmentsthereof, and polynucleotides encoding said polypeptides;

ANH0459C (SEQ ID NO 34): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 1-44, 153-176, 222-245,polypeptide fragments thereof, and polynucleotides encoding saidpolypeptides;

ANH0769 (SEQ ID NO 38): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 1-311, polypeptide fragmentsthereof, and polynucleotides encoding said polypeptides;

ANH0658 (SEQ ID NO 40): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 4-19, polypeptide fragmentsthereof, and polynucleotides encoding said polypeptides;

ANH0668 (SEQ ID NO 42): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 129-130, polypeptidefragments thereof, and polynucleotides encoding said polypeptides;

ANH0757 (SEQ ID NO 44): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 1-448, 448-449, 532-639,polypeptide fragments thereof, and polynucleotides encoding saidpolypeptides;

ANH0687A (SEQ ID NO 46): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 99-100, 283-314, polypeptidefragments thereof, and polynucleotides encoding said polypeptides;

ANH0687B (SEQ ID NO 48): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 490-491, polypeptidefragments thereof, and polynucleotides encoding said polypeptides;

ANH0693 (SEQ ID NO 50): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 1-91, 92-268, polypeptidefragments thereof, and polynucleotides encoding said polypeptides;

ANH0316 (SEQ ID NO 58): polypeptides comprising, consisting of, orconsisting essentially of about amino acids 1-25, 25-26, 115-116, 1-116,polypeptide fragments thereof, and polynucleotides encoding saidpolypeptides.

Nucleic Acids

A mammalian polynucleotide, or fragment thereof, of the presentinvention is a polynucleotide having a nucleotide sequence obtainablefrom a natural source. When the species name is used, e.g., humanANH0316, it indicates that the polynucleotide or polypeptide isobtainable from a natural source. It therefore includesnaturally-occurring normal, naturally-occurring mutant, andnaturally-occurring polymorphic alleles (e.g., SNPs),differentially-spliced transcripts, splice-variants, etc. By the term“naturally-occurring,” it is meant that the polynucleotide is obtainablefrom a natural source, e.g., animal tissue and cells, body fluids,tissue culture cells, forensic samples. Natural sources include, e.g.,living cells obtained from tissues and whole organisms, tumors, culturedcell lines, including primary and immortalized cell lines.Naturally-occurring mutations can include deletions (e.g., a truncatedamino- or carboxy-terminus), substitutions, inversions, or additions ofnucleotide sequence. These genes can be detected and isolated bypolynucleotide hybridization according to methods which one skilled inthe art would know, e.g., as discussed below.

A polynucleotide according to the present invention can be obtained froma variety of different sources. It can be obtained from DNA or RNA, suchas polyadenylated mRNA or total RNA, e.g., isolated from tissues, cells,or whole organism. The polynucleotide can be obtained directly from DNAor RNA, from a cDNA library, from a genomic library, etc. Thepolynucleotide can be obtained from a cell or tissue (e.g., from anembryonic or adult tissues) at a particular stage of development, havinga desired genotype, phenotype, disease status, etc. A polynucleotidewhich “codes without interruption” refers to a polynucleotide having acontinuous open reading frame (“ORF”) as compared to an ORF which isinterrupted by introns or other noncoding sequences.

Polynucleotides and polypeptides (including any part of thepolynucleotides listed in Tables 1-3) can be excluded as compositionsfrom the present invention if, e.g., listed in a publicly availabledatabases on the day this application was filed and/or disclosed in apatent application having an earlier filing or priority date than thisapplication and/or conceived and/or reduced to practice earlier than apolynucleotide in this application.

As described herein, the phrase “an isolated polynucleotide which is SEQID NO,” or “an isolated polynucleotide which is selected from SEQ IDNO,” refers to an isolated nucleic acid molecule from which the recitedsequence was derived (e.g., a cDNA derived from mRNA; cDNA derived fromgenomic DNA). Because of sequencing errors, typographical errors, etc.,the actual naturally-occurring sequence may differ from a SEQ ID listedherein. Thus, the phrase indicates the specific molecule from which thesequence was derived, rather than a molecule having that exact recitednucleotide sequence, analogously to how a culture depository numberrefers to a specific cloned fragment in a cryotube.

As explained in more detail below, a polynucleotide sequence of theinvention can contain the complete sequence as shown in SEQ ID NO 1-58,degenerate sequences thereof, anti-sense, muteins thereof, genescomprising said sequences, full-length cDNAs comprising said sequences,complete genomic sequences, fragments thereof, homologs, primers,nucleic acid molecules which hybridize thereto, derivatives thereof,etc.

Genomic

The present invention also relates genomic DNA from which thepolynucleotides of the present invention can be derived. A genomic DNAcoding for a human, mouse, or other mammalian polynucleotide, can beobtained routinely, for example, by screening a genomic library (e.g., aYAC library) with a polynucleotide of the present invention, or bysearching nucleotide databases, such as GenBank and EMBL, for matches.Promoter and other regulatory regions (including both 5′ and 3′ regions,as well introns) can be identified upstream of coding and expressedRNAs, and assayed routinely for activity, e.g., by joining to a reportergene (e.g., CAT, GFP, alkaline phosphatase, luciferase, galatosidase). Apromoter obtained from a polynucleotide of the present invention can beused, e.g., in gene therapy to obtain tissue-specific expression of aheterologous gene (e.g., coding for a therapeutic product or cytotoxin).5′ and/or 3′ sequences can also be used to modulate stability of anucleic acid, regulate its translation and/or transcription, etc.

Constructs

A polynucleotide of the present invention can comprise additionalpolynucleotide sequences, e.g., sequences to enhance expression,detection, uptake, cataloging, tagging, etc. A polynucleotide caninclude only coding sequence; a coding sequence and additionalnon-naturally occurring or heterologous coding sequence (e.g., sequencescoding for leader, signal, secretory, targeting, enzymatic, fluorescent,antibiotic resistance, and other functional or diagnostic peptides);coding sequences and non-coding sequences, e.g., untranslated sequencesat either a 5′ or 3′ end, or dispersed in the coding sequence, e.g.,introns.

A polynucleotide according to the present invention also can comprise anexpression control sequence operably linked to a polynucleotide asdescribed above. The phrase “expression control sequence” means apolynucleotide sequence that regulates expression of a polypeptide codedfor by a polynucleotide to which it is functionally (“operably”) linked.Expression can be regulated at the level of the mRNA or polypeptide.Thus, the expression control sequence includes mRNA-related elements andprotein-related elements. Such elements include promoters, enhancers(viral or cellular), ribosome binding sequences, transcriptionalterminators, etc. An expression control sequence is operably linked to anucleotide coding sequence when the expression control sequence ispositioned in such a manner to effect or achieve expression of thecoding sequence. For example, when a promoter is operably linked 5′ to acoding sequence, expression of the coding sequence is driven by thepromoter. Expression control sequences can include an initiation codonand additional nucleotides to place a partial nucleotide sequence of thepresent invention in-frame in order to produce a polypeptide (e.g., pETvectors from Promega have been designed to permit a molecule to beinserted into all three reading frames to identify the one that resultsin polypeptide expression). Expression control sequences can beheterologous or endogenous to the normal gene.

A polynucleotide of the present invention can also comprise nucleic acidvector sequences, e.g., for cloning, expression, amplification,selection, etc. Any effective vector can be used. A vector is, e.g., apolynucleotide molecule which can replicate autonomously in a host cell,e.g., containing an origin of replication. Vectors can be useful toperform manipulations, to propagate, and/or obtain large quantities ofthe recombinant molecule in a desired host. A skilled worker can selecta vector depending on the purpose desired, e.g., to propagate therecombinant molecule in bacteria, yeast, insect, or mammalian cells. Thefollowing vectors are provided by way of example. Bacterial: pQE70,pQE60, pQE-9 (Qiagen), pBS, pD10, Phagescript, phiX174, pBK Phagemid,pNH8A, pNH16a, pNH18Z, pNH46A (Stratagene); Bluescript KS+II(Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR54 0, pRIT5 (Pharmacia).Eukaryotic: PWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, PBPV,PMSG, pSVL (Pharmacia), pCR2.1/TOPO, pCRII/TOPO, pCR4/TOPO, pTrcHisB,pCMV6-XL4, etc. However, any other vector, e.g., plasmids, viruses, orparts thereof, may be used as long as they are replicable and viable inthe desired host. The vector can also comprise sequences which enable itto replicate in the host whose genome is to be modified.

Hybridization

Polynucleotide hybridization, as discussed in more detail below, isuseful in a variety of applications, including, in gene detectionmethods, for identifying mutations, for making mutations, to identifyhomologs in the same and different species, to identify related membersof the same gene family, in diagnostic and prognostic assays, intherapeutic applications (e.g., where an antisense polynucleotide isused to inhibit expression), etc.

The ability of two single-stranded polynucleotide preparations tohybridize together is a measure of their nucleotide sequencecomplementarity, e.g., base-pairing between nucleotides, such as A-T,G-C, etc. The invention thus also relates to polynucleotides, and theircomplements, which hybridize to a polynucleotide comprising a nucleotidesequence as set forth in SEQ ID NO 1-58 and genomic sequences thereof. Anucleotide sequence hybridizing to the latter sequence will have acomplementary polynucleotide strand, or act as a template for one in thepresence of a polymerase (i.e., an appropriate polynucleotidesynthesizing enzyme). The present invention includes both strands ofpolynucleotide, e.g., a sense strand and an anti-sense strand.

Hybridization conditions can be chosen to select polynucleotides whichhave a desired amount of nucleotide complementarity with the nucleotidesequences set forth in SEQ ID NO 1-58 and genomic sequences thereof. Apolynucleotide capable of hybridizing to such sequence, preferably,possesses, e.g., about 70%, 75%, 80%, 85%, 87%, 90%, 92%, 95%, 97%, 99%,or 100% complementarity, between the sequences. The present inventionparticularly relates to polynucleotide sequences which hybridize to thenucleotide sequences set forth in SEQ ID NO 1-58 or genomic sequencesthereof, under low or high stringency conditions. These conditions canbe used, e.g., to select corresponding homologs in non-human species.

Polynucleotides which hybridize to polynucleotides of the presentinvention can be selected in various ways. Filter-type blots (i.e.,matrices containing polynucleotide, such as nitrocellulose), glasschips, and other matrices and substrates comprising polynucleotides(short or long) of interest, can be incubated in a prehybridizationsolution (e.g., 6×SSC, 0.5% SDS, 100 μg/ml denatured salmon sperm DNA,5× Denhardt's solution, and 50% formamide), at 22-68° C., overnight, andthen hybridized with a detectable polynucleotide probe under conditionsappropriate to achieve the desired stringency. In general, when highhomology or sequence identity is desired, a high temperature can be used(e.g., 65° C.). As the homology drops, lower washing temperatures areused. For salt concentrations, the lower the salt concentration, thehigher the stringency. The length of the probe is another consideration.Very short probes (e.g., less than 100 base pairs) are washed at lowertemperatures, even if the homology is high. With short probes, formamidecan be omitted. See, e.g., Current Protocols in Molecular Biology,Chapter 6, Screening of Recombinant Libraries; Sambrook et al.,Molecular Cloning, 1989, Chapter 9.

For instance, high stringency conditions can be achieved by incubatingthe blot overnight (e.g., at least 12 hours) with a long polynucleotideprobe in a hybridization solution containing, e.g., about 5×SSC, 0.5%SDS, 100 μg/ml denatured salmon sperm DNA and 50% formamide, at 42° C.Blots can be washed at high stringency conditions that allow, e.g., forless than 5% bp mismatch (e.g., wash twice in 0.1% SSC and 0.1% SDS for30 min at 65° C.), i.e., selecting sequences having 95% or greatersequence identity.

Other non-limiting examples of high stringency conditions includes afinal wash at 65° C. in aqueous buffer containing 30 mM NaCl and 0.5%SDS. Another example of high stringent conditions is hybridization in 7%SDS, 0.5 M NaPO₄, pH 7, 1 mM EDTA at 50° C., e.g., overnight, followedby one or more washes with a 1% SDS solution at 42° C. Whereas highstringency washes can allow for less than 5% mismatch, reduced or lowstringency conditions can permit up to 20% nucleotide mismatch.Hybridization at low stringency can be accomplished as above, but usinglower formamide conditions, lower temperatures and/or lower saltconcentrations, as well as longer periods of incubation time.

Hybridization can also be based on a calculation of melting temperature(Tm) of the hybrid formed between the probe and its target, as describedin Sambrook et al. Generally, the temperature Tm at which a shortoligonucleotide (containing 18 nucleotides or fewer) will melt from itstarget sequence is given by the following equation: Tm=(number of A'sand T's)×2° C.+(number of C's and G's)×4° C. For longer molecules,Tm=81.5+16.6 log₁₀[Na⁺]+0.41(% GC)−600/N where [Na⁺] is the molarconcentration of sodium ions, % GC is the percentage of GC base pairs inthe probe, and N is the length. Hybridization can be carried out atseveral degrees below this temperature to ensure that the probe andtarget can hybridize. Mismatches can be allowed for by lowering thetemperature even further.

Stringent conditions can be selected to isolate sequences, and theircomplements, which have, e.g., at least about 90%, 95%, or 97%,nucleotide complementarity between the probe (e.g., a shortpolynucleotide of SEQ ID NO 1-58 or genomic sequences thereof) and atarget polynucleotide.

Other homologs of polynucleotides of the present invention can beobtained from mammalian and non-mammalian sources according to variousmethods. For example, hybridization with a polynucleotide can beemployed to select homologs, e.g., as described in Sambrook et al.,Molecular Cloning, Chapter 11, 1989. Such homologs can have varyingamounts of nucleotide and amino acid sequence identity and similarity tosuch polynucleotides of the present invention. Mammalian organismsinclude, e.g., mice, rats, monkeys, pigs, cows, etc. Non-mammalianorganisms include, e.g., vertebrates, invertebrates, zebra fish,chicken, Drosophila, C. elegans, Xenopus, yeast such as S. pombe, S.cerevisiae, roundworms, prokaryotes, plants, Arabidopsis, artemia,viruses, etc.

Alignment

Alignments can be accomplished by using any effective algorithm. Forpairwise alignments of DNA sequences, the methods described byWilbur-Lipman (e.g., Wilbur and Lipman, Proc. Natl. 4cad. Sci.,80:726-730, 1983) or Martinez/Needleman-Wunsch (e.g., Martinez, NucleicAcid Res., 11:4629-4634, 1983) can be used. For instance, if theMartinez/Needleman-Wunsch DNA alignment is applied, the minimum matchcan be set at 9, gap penalty at 1.10, and gap length penalty at 0.33.The results can be calculated as a similarity index, equal to the sum ofthe matching residues divided by the sum of all residues and gapcharacters, and then multiplied by 100 to express as a percent.Similarity index for related genes at the nucleotide level in accordancewith the present invention can be greater than 70%, 80%, 85%, 90%, 95%,99%, or more. Pairs of protein sequences can be aligned by theLipman-Pearson method (e.g., Lipman and Pearson, Science, 227:1435-1441,1985) with k-tuple set at 2, gap penalty set at 4, and gap lengthpenalty set at 12. Results can be expressed as percent similarity index,where related genes at the amino acid level in accordance with thepresent invention can be greater than 65%, 70%, 75%, 80%, 85%, 90%, 95%,99%, or more. Various commercial and free sources of alignment programsare available, e.g., MegAlign by DNA Star, BLAST (National Center forBiotechnology Information), BCM (Baylor College of Medicine) Launcher,etc. BLAST can be used to calculate amino acid sequence identity, aminoacid sequence homology, and nucleotide sequence identity. Thesecalculations can be made along the entire length of each of the targetsequences which are to be compared.

After two sequences have been aligned, a “percent sequence identity” canbe determined. For these purposes, it is convenient to refer to aReference Sequence and a Compared Sequence, where the Compared Sequenceis compared to the Reference Sequence. Percent sequence identity can bedetermined according to the following formula: Percent Identity=100[1−(C/R)], wherein C is the number of differences between the ReferenceSequence and the Compared Sequence over the length of alignment betweenthe Reference Sequence and the Compared Sequence where (i) each base oramino acid in the Reference Sequence that does not have a correspondingaligned base or amino acid in the Compared Sequence, (ii) each gap inthe Reference Sequence, (iii) each aligned base or amino acid in theReference Sequence that is different from an aligned base or amino acidin the Compared Sequence, constitutes a difference; and R is the numberof bases or amino acids in the Reference Sequence over the length of thealignment with the Compared Sequence with any gap created in theReference Sequence also being counted as a base or amino acid.

Percent sequence identity can also be determined by other conventionalmethods, e.g., as described in Altschul et al., Bull. Math. Bio. 48:603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA89:10915-10919, 1992.

Specific Polynucleotide Probes

A polynucleotide of the present invention can comprise any continuousnucleotide sequence of SEQ ID NO 1-58, sequences which share sequenceidentity thereto, or complements thereof. The term “probe” refers to anysubstance that can be used to detect, identify, isolate, etc., anothersubstance. A polynucleotide probe is comprised of nucleic acid can beused to detect, identify, etc., other nucleic acids, such as DNA andRNA.

These polynucleotides can be of any desired size that is effective toachieve the specificity desired. For example, a probe can be from about7 or 8 nucleotides to several thousand nucleotides, depending upon itsuse and purpose. For instance, a probe used as a primer PCR can beshorter than a probe used in an ordered array of polynucleotide probes.Probe sizes vary, and the invention is not limited in any way by theirsize, e.g., probes can be from about 7-2000 nucleotides, 7-1000, 8-700,8-600, 8-500, 8400, 8-300, 8-150, 8-100, 8-75, 7-50, 10-25, 14-16, atleast about 8, at least about 10, at least about 15, at least about 25,etc. The polynucleotides can have non-naturally-occurring nucleotides,e.g., inosine, AZT, 3TC, etc. The polynucleotides can have 100% sequenceidentity or complementarity to a sequence of SEQ ID NO 1-58, or it canhave mismatches or nucleotide substitutions, e.g., 1, 2, 3, 4, or 5substitutions. The probes can be single-stranded or double-stranded.

In accordance with the present invention, a polynucleotide can bepresent in a kit, where the kit includes, e.g., one or morepolynucleotides, a desired buffer (e.g., phosphate, tris, etc.),detection compositions, RNA or cDNA from different tissues to be used ascontrols, libraries, etc. The polynucleotide can be labeled orunlabeled, with radioactive or non-radioactive labels as known in theart. Kits can comprise one or more pairs of polynucleotides foramplifying nucleic acids specific for angiogenesis genes of the presentinvention, e.g., comprising a forward and reverse primer effective inPCR. These include both sense and anti-sense orientations. For instance,in PCR-based methods (such as RT-PCR), a pair of primers are typicallyused, one having a sense sequence and the other having an antisensesequence.

Another aspect of the present invention is a nucleotide sequence that isspecific to, or for, a selective polynucleotide. The phrases “specificfor” or “specific to” a polynucleotide have a functional meaning thatthe polynucleotide can be used to identify the presence of one or moretarget genes in a sample and distinguish them from non-target genes. Itis specific in the sense that it can be used to detect polynucleotidesabove background noise (“non-specific binding”). A specific sequence isa defined order of nucleotides (or amino acid sequences, if it is apolypeptide sequence) which occurs in the polynucleotide, e.g., in thenucleotide sequences of SEQ ID NO 1-58, and which is characteristic ofthat target sequence, and substantially no non-target sequences. A probeor mixture of probes can comprise a sequence or sequences that arespecific to a plurality of target sequences, e.g., where the sequence isa consensus sequence, a functional domain, etc., e.g., capable ofrecognizing a family of related genes. Such sequences can be used asprobes in any of the methods described herein or incorporated byreference. Both sense and antisense nucleotide sequences are included. Aspecific polynucleotide according to the present invention can bedetermined routinely.

A polynucleotide comprising a specific sequence can be used as ahybridization probe to identity the presence of, e.g., human or mousepolynucleotide, in a sample comprising a mixture of polynucleotides,e.g., on a Northern blot. Hybridization can be performed under highstringent conditions (see, above) to select polynucleotides (and theircomplements which can contain the coding sequence) having at least 90%,95%, 99%, etc., identity (i.e., complementarity) to the probe, but lessstringent conditions can also be used. A specific polynucleotidesequence can also be fused in-frame, at either its 5′ or 3′ end, tovarious nucleotide sequences as mentioned throughout the patent,including coding sequences for enzymes, detectable markers, GFP, etc,expression control sequences, etc.

A polynucleotide probe, especially one that is specific to apolynucleotide of the present invention, can be used in gene detectionand hybridization methods as already described. In one embodiment, aspecific polynucleotide probe can be used to detect whether a particulartissue or cell-type is present in a target sample. To carry out such amethod, a selective polynucleotide can be chosen which is characteristicof the desired target tissue. Such polynucleotide is preferably chosenso that it is expressed or displayed in the target tissue, but not inother tissues which are present in the sample. For instance, ifdetection of vascular is desired, it may not matter whether theselective polynucleotide is expressed in other tissues, as long as it isnot expressed in cells normally present in blood, e.g., peripheral bloodmononuclear cells. Starting from the selective polynucleotide, aspecific polynucleotide probe can be designed which hybridizes (ifhybridization is the basis of the assay) under the hybridizationconditions to the selective polynucleotide, whereby the presence of theselective polynucleotide can be determined.

Probes which are specific for polynucleotides of the present inventioncan also be prepared using involve transcription-based systems, e.g.,incorporating an RNA polymerase promoter into a selective polynucleotideof the present invention, and then transcribing anti-sense RNA using thepolynucleotide as a template. See, e.g., U.S. Pat. No. 5,545,522.

Polynucleotide Composition

A polynucleotide according to the present invention can comprise, e.g.,DNA, RNA, synthetic polynucleotide, peptide polynucleotide, modifiednucleotides, dsDNA, ssDNA, ssRNA, dsRNA, and mixtures thereof Apolynucleotide can be single- or double-stranded, triplex, DNA:RNA,duplexes, comprise hairpins, and other secondary structures, etc.Nucleotides comprising a polynucleotide can be joined via various knownlinkages, e.g., ester, sulfamate, sulfamide, phosphorothioate,phosphoramidate, methylphosphonate, carbamate, etc., depending on thedesired purpose, e.g., resistance to nucleases, such as RNAse H,improved in vivo stability, etc. See, e.g., U.S. Pat. No. 5,378,825. Anydesired nucleotide or nucleotide analog can be incorporated, e.g.,6-mercaptoguanine, 8-oxo-guanine, etc.

Various modifications can be made to the polynucleotides, such asattaching detectable markers (avidin, biotin, radioactive elements,fluorescent tags and dyes, energy transfer labels, energy-emittinglabels, binding partners, etc.) or moieties which improve hybridization,detection, and/or stability. The polynucleotides can also be attached tosolid supports, e.g., nitrocellulose, magnetic or paramagneticmicrospheres (e.g., as described in U.S. Pat. No. 5,411,863; U.S. Pat.No. 5,543,289; for instance, comprising ferromagnetic, supermagnetic,paramagnetic, superparamagnetic, iron oxide and polysaccharide), nylon,agarose, diazotized cellulose, latex solid microspheres,polyacrylamides, etc., according to a desired method. See, e.g., U.S.Pat. Nos. 5,470,967, 5,476,925, and 5,478,893.

Polynucleotide according to the present invention can be labeledaccording to any desired method. The polynucleotide can be labeled usingradioactive tracers such as ³²P, ³⁵S, ³H, or ¹⁴C, to mention somecommonly used tracers. The radioactive labeling can be carried outaccording to any method, such as, for example, terminal labeling at the3′ or 5′ end using a radiolabeled nucleotide, polynucleotide kinase(with or without dephosphorylation with a phosphatase) or a ligase(depending on the end to be labeled). A non-radioactive labeling canalso be used, combining a polynucleotide of the present invention withresidues having immunological properties (antigens, haptens), a specificaffinity for certain reagents (ligands), properties enabling detectableenzyme reactions to be completed (enzymes or coenzymes, enzymesubstrates, or other substances involved in an enzymatic reaction), orcharacteristic physical properties, such as fluorescence or the emissionor absorption of light at a desired wavelength, etc.

Nucleic Acid Detection Methods

Another aspect of the present invention relates to methods and processesfor detecting angiogenesis genes of the present invention. Detectionmethods have a variety of applications, including for diagnostic,prognostic, forensic, and research applications. To accomplish genedetection, a polynucleotide in accordance with the present invention canbe used as a “probe.” The term “probe” or “polynucleotide probe” has itscustomary meaning in the art, e.g., a polynucleotide which is effectiveto identify (e.g., by hybridization), when used in an appropriateprocess, the presence of a target polynucleotide to which it isdesigned. Identification can involve simply determining presence orabsence, or it can be quantitative, e.g., in assessing amounts of a geneor gene transcript present in a sample. Probes can be useful in avariety of ways, such as for diagnostic purposes, to identify homologs,and to detect, quantitate, or isolate a polynucleotide of the presentinvention in a test sample.

Assays can be utilized which permit quantification and/orpresence/absence detection of a target nucleic acid in a sample. Assayscan be performed at the single-cell level, or in a sample comprisingmany cells, where the assay is “averaging” expression over the entirecollection of cells and tissue present in the sample. Any suitable assayformat can be used, including, but not limited to, e.g., Southern blotanalysis, Northern blot analysis, polymerase chain reaction (“PCR”)(e.g., Saiki et al., Science, 241:53, 1988; U.S. Pat. Nos. 4,683,195,4,683,202, and 6,040,166; PCR Protocols: A Guide to Methods andApplications, Innis et al., eds., Academic Press, New York, 1990),reverse transcriptase polymerase chain reaction (“RT-PCR”), anchoredPCR, rapid amplification of cDNA ends (“RACE”) (e.g., Schaefer in GeneCloning and Analysis: Current Innovations, Pages 99-115, 1997), ligasechain reaction (“LCR”) (EP 320 308), one-sided PCR (Ohara et al., Proc.Natl. Acad. Sci., 86:5673-5677, 1989), indexing methods (e.g., U.S. Pat.No. 5,508,169), in situ hybridization, differential display (e.g., Lianget al., Nucl. Acid. Res., 21:3269-3275, 1993; U.S. Pat. Nos. 5,262,311,5,599,672 and 5,965,409; WO97/18454; Prashar and Weissman, Proc. Natl.Acad. Sci., 93:659-663, and U.S. Pat. Nos. 6,010,850 and 5,712,126;Welsh et al., Nucleic Acid Res., 20:4965-4970, 1992, and U.S. Pat. No.5,487,985) and other RNA fingerprinting techniques, nucleic acidsequence based amplification (“NASBA”) and other transcription basedamplification systems (e.g., U.S. Pat. Nos. 5,409,818 and 5,554,527; WO88/10315), polynucleotide arrays (e.g., U.S. Pat. Nos. 5,143,854,5,424,186; 5,700,637, 5,874,219, and 6,054,270; PCT WO 92/10092; PCT WO90/15070), Qbeta Replicase (PCT/US87/00880), Strand DisplacementAmplification (“SDA”), Repair Chain Reaction (“RCR”), nucleaseprotection assays, subtraction-based methods, Rapid-Scan™, etc.Additional useful methods include, but are not limited to, e.g.,template-based amplification methods, competitive PCR (e.g., U.S. Pat.No. 5,747,251), redox-based assays (e.g., U.S. Pat. No. 5,871,918),Taqman-based assays (e.g., Holland et al., Proc. Natl. Acad, Sci.,88:7276-7280, 1991; U.S. Pat. Nos. 5,210,015 and 5,994,063), real-timefluorescence-based monitoring (e.g., U.S. Pat. No. 5,928,907), molecularenergy transfer labels (e.g., U.S. Pat. Nos. 5,348,853, 5,532,129,5,565,322, 6,030,787, and 6,117,635; Tyagi and Kramer, Nature Biotech.,14:303-309, 1996). Any method suitable for single cell analysis of geneor protein expression can be used, including in situ hybridization,immunocytochemistry, MACS, FACS, flow cytometry, etc. For single cellassays, expression products can be measured using antibodies, PCR, orother types of nucleic acid amplification (e.g., Brady et al., MethodsMol. & Cell. Biol. 2, 17-25, 1990; Eberwine et al., 1992, Proc. Natl.Acad. Sci., 89, 3010-3014, 1992; U.S. Pat. No. 5,723,290). These andother methods can be carried out conventionally, e.g., as described inthe mentioned publications.

Many of such methods may require that the polynucleotide is labeled, orcomprises a particular nucleotide type useful for detection. The presentinvention includes such modified polynucleotides that are necessary tocarry out such methods. Thus, polynucleotides can be DNA, RNA, DNA:RNAhybrids, PNA, etc., and can comprise any modification or substituentwhich is effective to achieve detection.

Detection can be desirable for a variety of different purposes,including research, diagnostic, prognostic, and forensic. For diagnosticpurposes, it may be desirable to identify the presence or quantity of apolynucleotide sequence in a sample, where the sample is obtained fromtissue, cells, body fluids, etc. In a preferred method as described inmore detail below, the present invention relates to a method ofdetecting a polynucleotide comprising, contacting a targetpolynucleotide in a test sample with a polynucleotide probe underconditions effective to achieve hybridization between the target andprobe; and detecting hybridization.

Any test sample in which it is desired to identify a polynucleotide orpolypeptide thereof can be used, including, e.g., blood, urine, saliva,stool (for extracting nucleic acid, see, e.g., U.S. Pat. No. 6,177,251),swabs comprising tissue, biopsied tissue, tissue sections, culturedcells, etc.

Detection can be accomplished in combination with polynucleotide probesfor other genes, e.g., genes which are expressed in other diseasestates, tissues, cells, such as brain, heart, kidney, spleen, thymus,liver, stomach, small intestine, colon, muscle, lung, testis, placenta,pituitary, thyroid, skin, adrenal gland, pancreas, salivary gland,uterus, ovary, prostate gland, peripheral blood cells (T-cells,lymphocytes, etc.), embryo, normal breast fat, adult and embryonic stemcells, specific cell-types, such as endothelial, epithelial, myocytes,adipose, luminal epithelial, basoepithelial, myoepithelial, stromalcells, etc.

Polynucleotides can be used in wide range of methods and compositions,including for detecting, diagnosing, staging, grading, assessing,prognosticating, etc. diseases and disorders associated withangiogenesis genes and polypeptides of the present invention, formonitoring or assessing therapeutic and/or preventative measures, inordered arrays, etc. Any method of detecting genes and polynucleotidesrepresented by SEQ ID NO. 1-58, and variations thereof, can be used;certainly, the present invention is not to be limited how such methodsare implemented.

Along these lines, the present invention relates to methods of detectingangiogenesis genes of the present invention in a sample comprisingnucleic acid. Such methods can comprise one or more the following stepsin any effective order, e.g., contacting said sample with apolynucleotide probe under conditions effective for said probe tohybridize specifically to nucleic acid in said sample, and detecting thepresence or absence of probe hybridized to nucleic acid in said sample,wherein said probe can be a polynucleotide selected from SEQ ID NO 1-58,a polynucleotide having, e.g., about 70%, 80%, 85%, 90%, 95%, 99%, ormore sequence identity thereto, effective or specific fragments thereof,or complements thereto. The detection method can be applied to anysample, e.g., cultured primary, secondary, or established cell lines,tissue biopsy, blood, urine, stool, cerebral spinal fluid, and otherbodily fluids, for any purpose.

Contacting the sample with probe can be carried out by any effectivemeans in any effective environment. It can be accomplished in a solid,liquid, frozen, gaseous, amorphous, solidified, coagulated, colloid,etc., mixtures thereof, matrix. For instance, a probe in an aqueousmedium can be contacted with a sample which is also in an aqueousmedium, or which is affixed to a solid matrix, or vice-versa.

Generally, as used throughout the specification, the term “effectiveconditions” means, e.g., the particular milieu in which the desiredeffect is achieved. Such a milieu, includes, e.g., appropriate buffers,oxidizing agents, reducing agents, pH, co-factors, temperature, ionconcentrations, suitable age and/or stage of cell (such as, inparticular part of the cell cycle, or at a particular stage whereparticular genes are being expressed) where cells are being used,culture conditions (including substrate, oxygen, carbon dioxide, etc.).When hybridization is the chosen means of achieving detection, the probeand sample can be combined such that the resulting conditions arefunctional for said probe to hybridize specifically to nucleic acid insaid sample.

The phrase “hybridize specifically” indicates that the hybridizationbetween single-stranded polynucleotides is based on nucleotide sequencecomplementarity. The effective conditions are selected such that theprobe hybridizes to a preselected and/or definite target nucleic acid inthe sample. For instance, if detection of a polynucleotide set forth inSEQ ID NO 1-58, or a polymorphism thereof, is desired, a probe can beselected which can hybridize to such target gene under high stringentconditions, without significant hybridization to other genes in thesample. To detect homologs of a polynucleotide set forth in SEQ D NO1-58, the effective hybridization conditions can be less stringent,and/or the probe can comprise codon degeneracy, such that a homolog isdetected in the sample.

As already mentioned, the methods can be carried out by any effectiveprocess, e.g., by Northern blot analysis, polymerase chain reaction(PCR), reverse transcriptase PCR, RACE PCR, in situ hybridization, etc.,as indicated above. When PCR based techniques are used, two or moreprobes are generally used. One probe can be specific for a definedsequence which is characteristic of a selective polynucleotide, but theother probe can be specific for the selective polynucleotide, orspecific for a more general sequence, e.g., a sequence such as polyAwhich is characteristic of mRNA, a sequence which is specific for apromoter, ribosome binding site, or other transcriptional features, aconsensus sequence (e.g., representing a functional domain). For theformer aspects, 5′ and 3′ probes (e.g., polyA, Kozak, etc.) arepreferred which are capable of specifically hybridizing to the ends oftranscripts. When PCR is utilized, the probes can also be referred to as“primers” in that they can prime a DNA polymerase reaction.

In addition to testing for the presence or absence of polynucleotides,the present invention also relates to determining the amounts at whichpolynucleotides of the present invention are expressed in sample anddetermining the differential expression of such polynucleotides insamples. Such methods can involve substantially the same steps asdescribed above for presence/absence detection, e.g., contacting withprobe, hybridizing, and detecting hybridized probe, but using morequantitative methods and/or comparisons to standards.

The amount of hybridization between the probe and target can bedetermined by any suitable methods, e.g., PCR, RT-PCR, RACE PCR,Northern blot, polynucleotide microarrays, Rapid-Scan, etc., andincludes both quantitative and qualitative measurements. For furtherdetails, see the hybridization methods described above and below.Determining by such hybridization whether the target is differentiallyexpressed (e.g., up-regulated or down-regulated) in the sample can alsobe accomplished by any effective means. For instance, the target'sexpression pattern in the sample can be compared to its pattern in aknown standard, such as in a normal tissue, or it can be compared toanother gene in the same sample. When a second sample is utilized forthe comparison, it can be a sample of normal tissue that is known not tocontain diseased cells. The comparison can be performed on samples whichcontain the same amount of RNA (such as polyadenylated RNA or totalRNA), or, on RNA extracted from the same amounts of starting tissue.Such a second sample can also be referred to as a control or standard.Hybridization can also be compared to a second target in the same tissuesample. Experiments can be performed that determine a ratio between thetarget nucleic acid and a second nucleic acid (a standard or control),e.g., in a normal tissue. When the ratio between the target and controlare substantially the same in a normal and sample, the sample isdetermined or diagnosed not to contain cells. However, if the ratio isdifferent between the normal and sample tissues, the sample isdetermined to contain cancer cells. The approaches can be combined, andone or more second samples, or second targets can be used. Any secondtarget nucleic acid can be used as a comparison, including“housekeeping” genes, such as beta-actin, alcohol dehydrogenase, or anyother gene whose expression does not vary depending upon the diseasestatus of the cell.

Methods of Identifying Polymorphisms, Mutations, etc., of AngiogenesisGenes of the Present Invention

Polynucleotides of the present invention can also be utilized toidentify mutant alleles, SNPs, gene rearrangements and modifications,and other polymorphisms of the wild-type gene. Mutant alleles,polymorphisms, SNPs, etc., can be identified and isolated from cancersthat are known, or suspected to have, a genetic component.Identification of such genes can be carried out routinely (see, abovefor more guidance), e.g., using PCR, hybridization techniques, directsequencing, mismatch reactions (see, e.g., above), RFLP analysis, SSCP(e.g., Orita et al., Proc. Natl. Acad. Sci., 86:2766, 1992), etc., wherea polynucleotide having a sequence selected from SEQ ID NO 1-58, andvariations thereof, are used as a probe. The selected mutant alleles,SNPs, polymorphisms, etc., can be used diagnostically to determinewhether a subject has, or is susceptible to a disorder associated with agene of the present invention, as well as to design therapies andpredict the outcome of the disorder. Methods involve, e.g., diagnosing adisorder associated with an angiogenesis polynucleotide or polypeptide,or determining susceptibility to a disorder, comprising, detecting thepresence of a mutation in a gene represented by a polynucleotideselected from SEQ ID NO 1-58, and variations thereof. The detecting canbe carried out by any effective method, e.g., obtaining cells from asubject, determining the gene sequence or structure of a target gene(using, e.g., mRNA, cDNA, genomic DNA, etc), comparing the sequence orstructure of the target gene to the structure of the normal gene,whereby a difference in sequence or structure indicates a mutation inthe gene in the subject. Polynucleotides can also be used to test formutations, SNPs, polymorphisms, etc., e.g., using mismatch DNA repairtechnology as described in U.S. Pat. No. 5,683,877; U.S. Pat. No.5,656,430; Wu et al., Proc. Natl. Acad. Sci., 89:8779-8783, 1992.

The present invention also relates to methods of detecting polymorphismsin angiogenesis genes of the present invention, comprising, e.g.,comparing the structure of: genomic DNA comprising all or part of anangiogenesis gene of the present invention, mRNA comprising all or partof an angiogenesis gene of the present invention, cDNA comprising all orpart of an angiogenesis gene of the present invention, or a polypeptidecomprising all or part of an angiogenesis gene of the present invention,with the structure of an angiogenesis gene of the present invention,e.g., as set forth in SEQ ID NO 1-58. The methods can be carried out ona sample from any source, e.g., cells, tissues, body fluids, blood,urine, stool, hair, egg, sperm, etc.

These methods can be implemented in many different ways. For example,“comparing the structure” steps include, but are not limited to,comparing restriction maps, nucleotide sequences, amino acid sequences,RFLPs, DNase sites, DNA methylation fingerprints (e.g., U.S. Pat. No.6,214,556), protein cleavage sites, molecular weights, electrophoreticmobilities, charges, ion mobility, etc., between a standard gene (e.g.,SEQ ID NO 1-58) and a polymorphism. The term “structure” can refer toany physical characteristics or configurations which can be used todistinguish between nucleic acids and polypeptides. The methods andinstruments used to accomplish the comparing step depends upon thephysical characteristics which are to be compared. Thus, varioustechniques are contemplated, including, e.g., sequencing machines (bothamino acid and polynucleotide), electrophoresis, mass spectrometer (U.S.Pat. Nos. 6,093,541, 6,002,127), liquid chromatography, HPLC, etc.

To carry out such methods, “all or part” of the gene or polypeptide canbe compared. For example, if nucleotide sequencing is utilized, theentire gene can be sequenced, including promoter, introns, and exons, oronly parts of it can be sequenced and compared, e.g., exon 1, exon 2,etc.

Mutagenesis

Mutated polynucleotide sequences of the present invention are useful forvarious purposes, e.g., to create mutations of the polypeptides theyencode, to identify functional regions of genomic DNA, to produce probesfor screening libraries, etc. Mutagenesis can be carried out routinelyaccording to any effective method, e.g., oligonucleotide-directed(Smith, M., Ann. Rev. Genet. 19:423-463, 1985), degenerateoligonucleotide-directed (Hill et al., Method Enzymology, 155:558-568,1987), region-specific (Myers et al., Science, 229:242-246, 1985;Derbyshire et al., Gene, 46:145, 1986; Ner et al., DNA, 7:127, 1988),linker-scanning (McKnight and Kingsbury, Science, 217:316-324, 1982),directed using PCR, recursive ensemble mutagenesis (Arkin and Yourvan,Proc. Natl. Acad. Sci., 89:7811-7815, 1992), random mutagenesis (e.g.,U.S. Pat. Nos. 5,096,815; 5,198,346; and 5,223,409), site-directedmutagenesis (e.g., Walder et al., Gene, 42:133, 1986; Bauer et al.,Gene, 37:73, 1985; Craik, Bio Techniques, January 1985, 12-19; Smith etal., Genetic Engineering: Principles and Methods, Plenum Press, 1981),phage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991;Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/06204), etc. Desired sequences can also be produced by the assemblyof target sequences using mutually priming oligonucleotides (Uhlmann,Gene, 71:29-40, 1988). For directed mutagenesis methods, analysis of thethree-dimensional structure of a polypeptide can be used to guide andfacilitate making mutants which effect polypeptide activity. Sites ofsubstrate-enzyme interaction or other biological activities can also bedetermined by analysis of crystal structure as determined by suchtechniques as nuclear magnetic resonance, crystallography orphotoaffinity labeling. See, for example, de Vos et al., Science255:306-312, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992;Wlodaver et al., FEBS Lett. 309:59-64, 1992.

In addition, libraries of angiogenesis genes and fragments thereof canbe used for screening and selection of variants. For instance, a libraryof coding sequences can be generated by treating a double-stranded DNAwith a nuclease under conditions where the nicking occurs, e.g., onlyonce per molecule, denaturing the double-stranded DNA, renaturing it tofor double-stranded DNA that can include sense/antisense pairs fromdifferent nicked products, removing single-stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting DNAs into an expression vecore. By this method, xpressionlibraries can be made comprising a “mutagenized” gene. The entire codingsequence or parts thereof can be used.

Polynucleotide Expression, Polypeptides Produced Thereby, andSpecific-Binding Partners Thereto.

A polynucleotide according to the present invention can be expressed ina variety of different systems, in vitro and in vivo, according to thedesired purpose. For example, a polynucleotide can be inserted into anexpression vector, introduced into a desired host, and cultured underconditions effective to achieve expression of a polypeptide coded for bythe polynucleotide, to search for specific binding partners. Effectiveconditions include any culture conditions which are suitable forachieving production of the polypeptide by the host cell, includingeffective temperatures, pH, medium, additives to the media in which thehost cell is cultured (e.g., additives which amplify or induceexpression such as butyrate, or methotrexate if the codingpolynucleotide is adjacent to a dhfr gene), cycloheximide, celldensities, culture dishes, etc. A polynucleotide can be introduced intothe cell by any effective method including, e.g., naked DNA, calciumphosphate precipitation, electroporation, injection, DEAE-Dextranmediated transfection, fusion with liposomes, association with agentswhich enhance its uptake into cells, viral transfection. A cell intowhich a polynucleotide of the present invention has been introduced is atransformed host cell. The polynucleotide can be extrachromosomal orintegrated into a chromosome(s) of the host cell. It can be stable ortransient. An expression vector is selected for its compatibility withthe host cell. Host cells include, mammalian cells, e.g., COS, CV1, BHK,CHO, HeLa, LTK, NIH 3T3, 293, endothelial, epithelial, muscle, embryonicand adult stem cells, ectodermal, mesenchymal, endodermal, neoplastic,blood, bovine CPAE (CCL-209), bovine FBHE (CRL-1395), human HUV-EC-C(CRL-1730), mouse SVEC4-10EHR1 (CRL-2161), mouse MS1 (CRL-2279), mouseMS1 VEGF (CRL-2460), insect cells, such as Sf9 (S. frugipeda) andDrosophila, bacteria, such as E. coli, Streptococcus, bacillus, yeast,such as Sacharomyces, S. cerevisiae, fungal cells, plant cells,embryonic or adult stem cells (e.g., mammalian, such as mouse or human).

Expression control sequences are similarly selected for hostcompatibility and a desired purpose, e.g., high copy number, highamounts, induction, amplification, controlled expression. Othersequences which can be employed include enhancers such as from SV40,CMV, RSV, inducible promoters, cell-type specific elements, or sequenceswhich allow selective or specific cell expression. Promoters that can beused to drive its expression, include, e.g., the endogenous promoter,MMTV, SV40, trp, lac, tac, or T7 promoters for bacterial hosts; or alphafactor, alcohol oxidase, or PGH promoters for yeast. RNA promoters canbe used to produced RNA transcripts, such as T7 or SP6. See, e.g.,Melton et al., Polynucleotide Res., 12(18):7035-7056, 1984; Dunn andStudier. J. Mol. Bio., 166:477-435, 1984; U.S. Pat. No. 5,891,636;Studier et al., Gene Expression Technology, Methods in Enzymology,85:60-89, 1987. In addition, as discussed above, translational signals(including in-frame insertions) can be included.

When a polynucleotide is expressed as a heterologous gene in atransfected cell line, the gene is introduced into a cell as describedabove, under effective conditions in which the gene is expressed. Theterm “heterologous” means that the gene has been introduced into thecell line by the “hand-of-man.” Introduction of a gene into a cell lineis discussed above. The transfected (or transformed) cell expressing thegene can be lysed or the cell line can be used intact.

For expression and other purposes, a polynucleotide can contain codonsfound in a naturally-occurring gene, transcript, or cDNA, for example,e.g., as set forth in SEQ ID NO 1-58, or it can contain degeneratecodons coding for the same amino acid sequences. For instance, it may bedesirable to change the codons in the sequence to optimize the sequencefor expression in a desired host. See, e.g., U.S. Pat. Nos. 5,567,600and 5,567,862.

A polypeptide according to the present invention can be recovered fromnatural sources, transformed host cells (culture medium or cells)according to the usual methods, including, detergent extraction (e.g.,non-ionic detergent, Triton X-100, CHAPS, octylglucoside, IgepalCA-630), ammonium sulfate or ethanol precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, hydroxyapatitechromatography, lectin chromatography, gel electrophoresis. Proteinrefolding steps can be used, as necessary, in completing theconfiguration of the mature protein. Finally, high performance liquidchromatography (HPLC) can be employed for purification steps. Anotherapproach is express the polypeptide recombinantly with an affinity tag(Flag epitope, HA epitope, myc epitope, 6×His, maltose binding protein,chitinase, etc) and then purify by anti-tag antibody-conjugated affinitychromatography.

The present invention also relates to polypeptides of angiogenesis genesof the present invention, e.g., an isolated human polypeptide comprisingor having the amino acid sequence set forth in SEQ ID NO 1-58, anisolated polypeptide comprising an amino acid sequence having at leastabout 98%, 99%, or more amino acid sequence identity to the amino acidsequence set forth in SEQ ID NO 1-58, and optionally having one or moreof its activities.

Fragments specific to these polypeptides can also used, e.g., to produceantibodies or other immune responses, as well as competitors, agonists,antagonists, and ligands. These fragments can be referred to as being“specific for” the targeted gene. The latter phrase, as already defined,indicates that the peptides are characteristic of the targeted gene, andthat the defined sequences are substantially absent from all otherprotein types. Such polypeptides can be of any size which is necessaryto confer specificity, e.g., 5, 8, 10, 12, 15, 20, etc.

The present invention also relates to antibodies, and otherspecific-binding partners, which are specific for polypeptides encodedby polynucleotides of the present invention. Antibodies, e.g.,polyclonal, monoclonal, recombinant, chimeric, humanized, single-chain,Fab, and fragments thereof, can be prepared according to any desiredmethod. See, also, screening recombinant immunoglobulin libraries (e.g.,Orlandi et al., Proc. Natl. Acad. Sci., 86:3833-3837, 1989; Huse et al.,Science, 256:1275-1281, 1989); in vitro stimulation of lymphocytepopulations; Winter and Milstein, Nature, 349: 293-299, 1991. Theantibodies can be IgM, IgG, subtypes, IgG2a, IgG1, etc. Antibodies, andimmune responses, can also be generated by administering naked DNA See,e.g., U.S. Pat. Nos. 5,703,055; 5,589,466; 5,580,859. Antibodies can beused from any source, including, goat, rabbit, mouse, chicken (e.g.,IgY; see, Duan, WO/029444 for methods of making antibodies in avianhosts, and harvesting the antibodies from the eggs). An antibodyspecific for a polypeptide means that the antibody recognizes a definedsequence of amino acids within or including the polypeptide. Otherspecific binding partners include, e.g., aptamers and PNA. Antibodiescan be prepared against specific epitopes or domains, e.g., asidentified above.

The preparation of polyclonal antibodies is well-known to those skilledin the art. See, for example, Green et al., Production of PolyclonalAntisera, in IMMUNOCHEMICAL PROTOCOLS (Manson, ed.), pages 1-5 (HumanaPress 1992); Coligan et al., Production of Polyclonal Antisera inRabbits, Rats, Mice and Hamsters, in CURRENT PROTOCOLS IN IMMUNOLOGY,section 2.4.1 (1992). The preparation of monoclonal antibodies likewiseis conventional. See, for example, Kohler & Milstein, Nature 256:495(1975); Coligan et al., sections 2.5.1-2.6.7; and Harlow et al.,ANTIBODIES: A LABORATORY MANUAL, page 726 (Cold Spring Harbor Pub.1988).

Antibodies can also be humanized, e.g., where they are to be usedtherapeutically. Humanized monoclonal antibodies are produced bytransferring mouse complementarity determining regions from heavy andlight variable chains of the mouse immunoglobulin into a human variabledomain, and then substituting human residues in the framework regions ofthe murine counterparts. The use of antibody components derived fromhumanized monoclonal antibodies obviates potential problems associatedwith the immunogenicity of murine constant regions. General techniquesfor cloning murine immunoglobulin variable domains are described, forexample, by Orlandi et al., Proc. Nat. Acad. Sci. USA 86:3833 (1989),which is hereby incorporated in its entirety by reference. Techniquesfor producing humanized monoclonal antibodies are described, forexample, in U.S. Pat. No. 6,054,297, Jones et al., Nature 321: 522(1986); Riechmann et al., Nature 332: 323 (1988); Verhoeyen et al.,Science 239: 1534 (1988); Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285 (1992); Sandhu, Crit. Rev. Biotech. 12: 437 (1992); and Singer etal., J. Immunol. 150: 2844 (1993).

Antibodies of the invention also may be derived from human antibodyfragments isolated from a combinatorial immunoglobulin library. See, forexample, Barbas et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY,VOL. 2, page 119 (1991); Winter et al., Ann. Rev. Immunol. 12: 433(1994). Cloning and expression vectors that are useful for producing ahuman immunoglobulin phage library can be obtained commercially, forexample, from STRATAGENE Cloning Systems (La Jolla, Calif.).

In addition, antibodies of the present invention may be derived from ahuman monoclonal antibody. Such antibodies are obtained from transgenicmice that have been “engineered” to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain loci are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous heavy and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens and can be usedto produce human antibody-secreting hybridomas. Methods for obtaininghuman antibodies from transgenic mice are described, e.g., in Green etal., Nature Genet. 7:13 (1994); Lonberg et al., Nature 368:856 (1994);and Taylor et al., Int. Immunol. 6:579 (1994).

Antibody fragments of the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ofnucleic acid encoding the fragment. Antibody fragments can be obtainedby pepsin or papain digestion of whole antibodies by conventionalmethods. For example, antibody fragments can be produced by enzymaticcleavage of antibodies with pepsin to provide a 5S fragment denotedF(ab′).sub.2. This fragment can be further cleaved using a thiolreducing agent, and optionally a blocking group for the sulfhydrylgroups resulting from cleavage of disulfide linkages, to produce 3.5SFab′ monovalent fragments. Alternatively, an enzymatic cleavage usingpepsin produces two monovalent Fab′ fragments and an Fc fragmentdirectly. These methods are described, for example, by Goldenberg, U.S.Pat. No. 4,036,945 and No. 4,331,647, and references contained therein.These patents are hereby incorporated in their entireties by reference.See also Nisoiihoff et al., Arch. Biochem. Biophys. 89:230 (1960);Porter, Biochem. J. 73:119 (1959); Edelman et al, METHODS IN ENZYMOLOGY,VOL. 1, page 422 (Academic Press 1967); and Coligan et al. at sections2.8.1-2.8.10 and 2.10.1-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques can alsobe used. For example, Fv fragments comprise an association of V.sub.Hand V.sub.L chains. This association may be noncovalent, as described inInbar et al., Proc. Nat'l Acad. Sci. USA 69:2659 (1972). Alternatively,the variable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde. See, e.g., Sandhu,supra. Preferably, the Fv fragments comprise V.sub.H and V.sub.L chainsconnected by a peptide linker. These single-chain antigen bindingproteins (sFv) are prepared by constructing a structural gene comprisingnucleic acid sequences encoding the V.sub.H and V.sub.L domainsconnected by an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cellsuch as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow etal., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 97(1991); Bird et al., Science 242:423-426 (1988); Ladneret al., U.S. Pat.No. 4,946,778; Pack et al., Bio/Technology 11: 1271-77 (1993); andSandhu, supra.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick et al.,METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 106 (1991).

The term “antibody” as used herein includes intact molecules as well asfragments thereof, such as Fab, F(ab′)₂, and Fv which are capable ofbinding to an epitopic determinant present in Bin1 polypeptide. Suchantibody fragments retain some ability to selectively bind with itsantigen or receptor. The term “epitope” refers to an antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics. Antibodies can be preparedagainst specific epitopes or polypeptide domains.

Antibodies which bind to polypeptides of the present invention can beprepared using an intact polypeptide or fragments containing smallpeptides of interest as the immunizing antigen. The polypeptide orpeptide used to immunize an animal can be conjugated to a carrierprotein, if desired. Such commonly used carriers which are chemicallycoupled to the immunizing peptide include keyhole limpet hemocyanin(KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.

Polyclonal or monoclonal antibodies can be further purified, forexample, by binding to and elution from a matrix to which thepolypeptide or a peptide to which the antibodies were raised is bound.Those of skill in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies (See for example, Coligan,et al., Unit 9, Current Protocols in Immunology, Wiley Interscience,1994, incorporated by reference).

Anti-idiotype technology can also be used to produce inventionmonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody.

Methods of Detecting Polypeptides

Polypeptides coded for genes of the present invention can be detected,visualized, determined, quantitated, etc. according to any effectivemethod useful methods include, e.g., but are not limited to,immunoassays, RIA (radioimmunassay), ELISA, (enzyme-linked-immunosorbentassay), immunoflourescence, flow cytometry, histology, electronmicroscopy, light microscopy, in situ assays, immunoprecipitation,Western blot, etc.

Immunoassays may be carried in liquid or on biological support. Forinstance, a sample (e.g., blood, stool, urine, cells, tissue, bodyfluids, etc.) can be brought in contact with and immobilized onto asolid phase support or carrier such as nitrocellulose, or other solidsupport that is capable of immobilizing cells, cell particles or solubleproteins. The support may then be washed with suitable buffers followedby treatment with the detectably labeled gene specific antibody. Thesolid phase support can then be washed with a buffer a second time toremove unbound antibody. The amount of bound label on solid support maythen be detected by conventional means.

A “solid phase support or carrier” includes any support capable ofbinding an antigen, antibody, or other specific binding partner.Supports or carriers include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, and magnetite. A support material can have anystructural or physical configuration. Thus, the support configurationmay be spherical, as in a bead, or cylindrical, as in the inside surfaceof a test tube, or the external surface of a rod. Alternatively, thesurface may be flat such as a sheet, test strip, etc. Preferred supportsinclude polystyrene beads

One of the many ways in which gene peptide-specific antibody can bedetectably labeled is by linking it to an enzyme and using it in anenzyme immunoassay (EIA). See, e.g., Voller, A., “The Enzyme LinkedImmunosorbent Assay (ELISA),” 1978, Diagnostic Horizons 2, 1-7,Microbiological Associates Quarterly Publication, Walkersville, Md.);Voller, A. et al., 1978, J. Clin. Pathol. 31, 507-520; Butler, J. E.,1981, Meth. Enzymol. 73, 482-523; Maggio, E. (ed.), 1980, EnzymeImmunoassay, CRC Press, Boca Raton, Fla. The enzyme which is bound tothe antibody will react with an appropriate substrate, preferably achromogenic substrate, in such a manner as to produce a chemical moietythat can be detected, for example, by spectrophotometric, fluorimetricor by visual means. Enzymes that can be used to detectably label theantibody include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha.-glycerophosphate, dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta.-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods that employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect peptides through the use ofa radioimmunoassay (RIA). See, e.g., Weintraub, B., Principles ofRadioimmunoassays, Seventh Training Course on Radioligand AssayTechniques, The Endocrine Society, March, 1986. The radioactive isotopecan be detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. Theantibody can also be detectably labeled using fluorescence emittingmetals such as those in the lanthanide series. These metals can beattached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples of usefulchemiluminescent labeling compounds are luminol, isoluminol, theromaticacridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

Diagnostic

The present invention also relates to methods and compositions fordiagnosing a vascular disorder, or determining susceptibility to adisorder, using polynucleotides, polypeptides, and specific-bindingpartners of the present invention to detect, assess, determine, etc.,genes of the present invention. In such methods, the gene can serve as amarker for the disorder, e.g., where the gene, when mutant, is a directcause of the disorder, where the gene is affected by another gene(s)which is directly responsible for the disorder, e.g., when the gene ispart of the same signaling pathway as the directly responsible gene;and, where the gene is chromosomally linked to the gene(s) directlyresponsible for the disorder, and segregates with it. Many othersituations are possible. To detect, assess, determine, etc., a probespecific for the gene can be employed as described above and below. Anymethod of detecting and/or assessing the gene can be used, includingdetecting expression of the gene using polynucleotides, antibodies, orother specific-binding partners.

The present invention relates to methods of diagnosing a vasculardisorder or a disorder associated with a gene of the present inventionor determining a subject's susceptibility to such disorder, comprising,e.g., assessing the expression of said gene (or polypeptide encodedthereby) in a tissue sample comprising tissue or cells suspected ofhaving the disorder (e.g., where the sample comprises vascular tissue).The phrase “diagnosing” indicates that it is determined whether thesample has the disorder. A “disorder” means, e.g., any abnormalcondition as in a disease or malady. “Determining a subject'ssusceptibility to a disease or disorder” indicates that the subject isassessed for whether s/he is predisposed to get such a disease ordisorder, where the predisposition is indicated by abnormal expressionof the gene (e.g., gene mutation, gene expression pattern is not normal,etc.). Predisposition or susceptibility to a disease may result when asuch disease is influenced by epigenetic, environmental, etc., factors.This includes prenatal screening where samples from the fetus or embryo(e.g., via amniocentesis or CV sampling) are analyzed for the expressionof the gene. Such diseases include, e.g., inflammatory diseases, such asrheumatoid arthritis, osteoarthritis, asthma, pulmonary fibrosis,age-related macular degeneration (ARMD), diabetic retinopathy, maculardegeneration, and retinopathy of prematurity (ROP), endometriosis,cancer, Coats' disease, peripheral retinal neovascularization,neovascular glaucoma, psoriasis, retrolental fibroplasias, angiofibroma,inflammation, etc. Examples of other diseases associated with genes ofthe present invention as shown in Table 2.

By the phrase “assessing expression of said gene,” it is meant that thefunctional status of the gene is evaluated. This includes, but is notlimited to, measuring expression levels of said gene, determining thegenomic structure of said gene, determining the mRNA structure oftranscripts from said gene, or measuring the expression levels ofpolypeptide coded for by said gene. Thus, the term “assessingexpression” includes evaluating the all aspects of the transcriptionaland translational machinery of the gene. For instance, if a promoterdefect causes, or is suspected of causing, the disorder, then a samplecan be evaluated (i.e., “assessed”) by looking (e.g., sequencing orrestriction mapping) at the promoter sequence in the gene, by detectingtranscription products (e.g., RNA), by detecting translation product(e.g., polypeptide). Any measure of whether the gene is functional canbe used, including, polypeptide, polynucleotide, and functional assaysfor the gene's biological activity.

In making the assessment, it can be useful to compare the results to anormal gene, e.g., a gene which is not associated with the disorder. Thenature of the comparison can be determined routinely, depending upon howthe assessing is accomplished. If, for example, the mRNA levels of asample is detected, then the mRNA levels of a normal can serve as acomparison, or a gene which is known not to be affected by the disorder.Methods of detecting mRNA are well known, and discussed above, e.g., butnot limited to, Northern blot analysis, polymerase chain reaction (PCR),reverse transcriptase PCR, RACE PCR, etc. Similarly, if polypeptideproduction is used to evaluate the gene, then the polypeptide in anormal tissue sample can be used as a comparison, or, polypeptide from adifferent gene whose expression is known not to be affected by thedisorder. These are only examples of how such a method could be carriedout.

Assessing the effects of therapeutic and preventative interventions(e.g., administration of a drug, chemotherapy, radiation, etc.) onvascular disorders or conditions is a major effort in drug discovery,clinical medicine, and pharmacogenomics. The evaluation of therapeuticand preventative measures, whether experimental or already in clinicaluse, has broad applicability, e.g., in clinical trials, for monitoringthe status of a patient, for analyzing and assessing animal models, andin any scenario involving cancer treatment and prevention. Analyzing theexpression profiles of polynucleotides of the present invention can beutilized as a parameter by which interventions are judged and measured.Treatment of a disorder can change the expression profile in some mannerwhich is prognostic or indicative of the drug's effect on it. Changes inthe profile can indicate, e.g., drug toxicity, return to a normal level,etc. Accordingly, the present invention also relates to methods ofmonitoring or assessing a therapeutic or preventative measure (e.g.,chemotherapy, radiation, anti-neoplastic drugs, antibodies, etc.) in asubject having a condition or disorder associated with angiogenesis,comprising, e.g., detecting the expression levels of a gene orpolypeptide of the present invention. A subject can be a cell-basedassay system, non-human animal model, human patient, etc. Detecting canbe accomplished as described for the methods above and below. By“therapeutic or preventative intervention,” it is meant, e.g., a drugadministered to a patient, surgery, radiation, chemotherapy, and othermeasures taken to prevent, treat, or diagnose a disorder.

Methods of Detecting Angiogenesis

The present invention also relates to detecting the presence and/orextent of blood vessels in a sample. The detected blood vessels can beestablished or pre-existing vessels, newly formed vessels, vessels inthe process of forming, or combinations thereof. A blood vessel includesany biological structure that conducts blood, including arteries, veins,capillaries, microvessels, vessel lumen, endothelial-lined sinuses, etc.These methods are useful for a variety of purposes. In cancer, forinstance, the extent of vascularization can be an important factor indetermining the clinical behavior of neoplastic cells. See, e.g.,Weidner et al., N. Engl. J. Med., 324:1-8, 1991. Thus, the presence andextent of blood vessels, including the angiogenic process itself, can beuseful for the diagnosis, prognosis, treatment, etc., of cancer andother neoplasms. Detection of vessels can also be utilized for thediagnosis, prognosis, treatment, of any diseases or conditionsassociated with vessel growth and production, to assess agents whichmodulate angiogenesis, to assess angiogenic gene therapy, etc.

An example of a method of detecting the presence or extent of bloodvessels in a sample is determining an angiogenic index of a tissue orcell sample comprising, e.g., assessing in a sample, the expressionlevels of a nucleic acid or polypeptide of the present invention,whereby said levels are indicative of the angiogenic index. By thephrase “angiogenic index,” it is meant the extent or degree ofvascularity of the tissue, e.g., the number or amount of blood vesselsin the sample of interest. Amounts of nucleic acid or polypeptide can beassessed (e.g., determined, detected, etc.) by any suitable method.There is no limitation on how detection is performed.

For instance, if nucleic acid is to be assessed, e.g., an mRNAcorresponding to a differentially-expressed gene, the methods fordetecting it, assessing its presence and/or amount, can be determined byany the methods mentioned above, e.g., nucleic acid based detectionmethods, such as Northern blot analysis, RT-PCR, RACE, differentialdisplay, NASBA and other transcription based amplification systems,polynucleotide arrays, etc. If RT-PCR is employed, cDNA can be preparedfrom the mRNA extracted from a sample of interest. Once the cDNA isobtained, PCR can be employed using oligonucleotide primer pairs thatare specific for a differentially-expressed gene. The specific probescan be of a single sequence, or they can be a combination of differentsequences. A polynucleotide array can also be used to assess nucleic,e.g., where the RNA of the sample of interest is labeled (e.g., using atranscription based amplification method, such as U.S. Pat. No.5,716,785) and then hybridized to probe fixed to a solid substrate.

Polypeptide detection can also be carried out by any available method,e.g., by Western blots, ELISA, dot blot, immunoprecipitation, RIA,immunohistochemistry, etc. For instance, a tissue section can beprepared and labeled with a specific antibody (indirect or direct),visualized with a microscope, and then the number of vessels in aparticular field of view counted, where staining with antibody is usedto identify and count the vessels. Amount of a polypeptide can bequantitated without visualization, e.g., by preparing a lysate of asample of interest, and then determining by ELISA or Western the amountof polypeptide per quantity of tissue. Again, there is no limitation onhow detection is performed.

In addition to assessing the angiogenic index using an antibody orpolynucleotide, other methods of determining tissue vascularity can beapplied. Tissue vascularity is typically determined by assessing thenumber and density of vesssels present in a given sample. For example,microvessel density (MVD) can be estimated by counting the number ofendothelial clusters in a high-power microscopic field, or detecting amarker specific for microvascular endothelium or other markers ofgrowing or established blood vessels, such as CD31 (also known asplatelet-endothelial cell adhesion molecule or PECAM). A CD31 antibodycan be employed in conventional immunohistological methods toimmunostain tissue sections as described by, e.g., Penfold et al., Br.J. Oral and Maxill. Surg., 34: 37-41; U.S. Pat. No. 6,017,949; Dellas etal., Gyn. Oncol., 67:27-33, 1997; and others.

In addition to the angiogenesis genes and polypeptides of the presentinvention, other genes and their corresponding products can be detected.For instance, it may be desired to detect a gene which is expressedubiquitously in the sample. A ubiquitously expressed gene, or productthereof, is present in all cell types, e.g., in about the same amount,e.g., beta-actin. Similarly, a gene or polypeptide that is expressedselectively in the tissue or cell of interest can be detected. Aselective gene or polypeptide is characteristic of the tissue orcell-type in which it is made. This can mean that it is expressed onlyin the tissue or cell, and in no other tissue- or cell-type, or it canmean that it is expressed preferentially, differentially, and moreabundantly (e.g., at least 5-fold, 10-fold, etc., or more) when comparedto other types. The expression of the ubiquitous or selective gene orgene product can be used as a control or reference marker to compare tothe expression of differentially-expression genes. Typically, expressionof the gene can be assessed by detecting mRNA produced from it. Othermarkers for blood vessels and angiogenesis can also be detected, such asangiogenesis-related genes or polypeptides. By the phrase“angiogenesis-related,” it is meant that it is associated with bloodvessels and therefore indicative of their presence. There are a numberof different genes and gene products that are angiogenesis-related,e.g., Vezf1 (e.g., Xiang et al., Dev. Bio., 206:123-141, 1999), VEGF,VEGF receptors (such as KDR/Flk-1), angiopoietin, Tie-1 and Tie-2 (e.g.,Sato et al., Nature, 376:70-74, 1995), PECAM-1 or CD31 (e.g., DAKO,Glostrup. Denmark), CD34, factor VIII-related antigen (e.g., Brustmannet al., Gyn. Oncol., 67:20-26, 1997).

Identifying Agent Methods

The present invention also relates to methods of identifying agents, andthe agents themselves, which modulate angiogenesis genes and thepolypeptides which encode them. These agents can be used to modulate thebiological activity of the polypeptide encoded for the gene, or thegene, itself. Agents which regulate the gene or its product are usefulin variety of different environments, including as medicinal agents totreat or prevent disorders associated with said genes, such asneovascularization in cancer, and as research reagents to modify thefunction of tissues and cell. In addition, the polypeptides these genesencode can interact with other proteins and binding partners (such asnucleic acids) which are present naturally in a cell, e.g., to formmulti-subunit functional assemblies and other complexes, that performspecific physiological functions in a cell.

Methods of identifying agents generally comprise steps in which an agentis placed in contact with the gene, transcription product, translationproduct, or other target, and then a determination is performed toassess whether the agent “modulates” the target. The specific methodutilized will depend upon a number of factors, including, e.g., thetarget (i.e., is it the gene or polypeptide encoded by it), theenvironment (e.g., in vitro or in vivo), the composition of the agent,etc.

For modulating the expression of a gene, a method can comprise, in anyeffective order, one or more of the following steps, e.g., contacting agene (e.g., in a cell population) with a test agent under conditionseffective for said test agent to modulate the expression of said gene,and determining whether said test agent modulates said gene. An agentcan modulate expression of the gene at any level, includingtranscription, translation, and/or perdurance of the nucleic acid (e.g.,degradation, stability, etc.) in the cell. For modulating the biologicalactivity of polypeptides, a method can comprise, in any effective order,one or more of the following steps, e.g., contacting a polypeptide(e.g., in a cell, lysate, or isolated) with a test agent underconditions effective for said test agent to modulate the biologicalactivity of said polypeptide, and determining whether said test agentmodulates said biological activity.

Contacting the gene or polypeptide with the test agent can beaccomplished by any suitable method and/or means that places the agentin a position to functionally control expression or biological activity.Functional control indicates that the agent can exert its physiologicaleffect through whatever mechanism it works. The choice of the methodand/or means can depend upon the nature of the agent and the conditionand type of environment in which the gene or polypeptide is presented,e.g., lysate, isolated, or in a cell population (such as, in vivo, invitro, organ explants, etc.). For instance, if the cell population is anin vitro cell culture, the agent can be contacted with the cells byadding it directly into the culture medium. If the agent cannot dissolvereadily in an aqueous medium, it can be incorporated into liposomes, oranother lipophilic carrier, and then administered to the cell culture.Contact can also be facilitated by incorporation of agent with carriersand delivery molecules and complexes, by injection, by infusion, etc.

After the agent has been administered in such a way that it can gainaccess to the gene or polypeptide, it can be determined whether the testagent modulates its expression or biological activity. Modulation can beof any type, quality, or quantity, e.g., increase, facilitate, enhance,up-regulate, stimulate, activate, amplify, augment, induce, decrease,down-regulate, diminish, lessen, reduce, etc. The modulatory quantitycan also encompass any value, e.g., 1%, 5%, 10%, 50%, 75%, 1-fold,2-fold, 5-fold, 10-fold, 100-fold, etc. To modulate gene expressionmeans, e.g., that the test agent has an effect on its expression, e.g.,to effect the amount of transcription, to effect RNA splicing, to effecttranslation of the RNA into polypeptide, to effect RNA or polypeptidestability, to effect polyadenylation or other processing of the RNA, toeffect post-transcriptional or post-translational processing, etc. Tomodulate biological activity means, e.g., that a functional activity ofthe polypeptide is changed in comparison to its normal activity in theabsence of the agent. This effect includes, increase, decrease, block,inhibit, enhance, etc.

A test agent can be of any molecular composition, e.g., chemicalcompounds, biomolecules, such as polypeptides, lipids, nucleic acids(e.g., antisense to a polynucleotide sequence selected from SEQ ID NO1-58), carbohydrates, antibodies, ribozymes, double-stranded RNA,aptamers, etc. For example, polypeptide fragments can be used tocompetitively inhibit polypeptides from binding to protein or DNA orfrom forming dimers. Antibodies can also be used to modulate thebiological activity a polypeptide in a lysate or other cell-free form.Antisense can also be used as test agents to modulate gene expression.

The present invention also relates to methods of identifying modulatorsof the genes and polypeptides of the present invention in a cellpopulation capable of forming blood vessels, comprising, one or more ofthe following steps in any effective order, e.g., contacting the cellpopulation with a test agent under conditions effective for said testagent to modulate its expression or biological activity. These methodsare useful, e.g., for drug discovery in identifying and confirming theangiogenic activity of agents, for identifying molecules in the normalpathway of angiogenesis, etc.

Any cell population capable of forming blood vessels can be utilized.Useful models, included those mentioned above, e.g., in vivoMatrigel-type assays, tumor neovascularization assays, CAM assays, BCEassays, migration assays, HUVEC growth inhibition assays, animal models(e.g., tumor growth in athymic mice), models involving hybrid cell andelectronic-based components, etc. Cells can include, e.g., endothelial,epithelial, muscle, embryonic and adult stem cells, ectodermal,mesenchymal, endodermal, neoplastic, blood, bovine CPAE (CCL-209),bovine FBHE (CRL-1395), human HUV-EC-C(CRL-1730), mouse SVEC4-10EHR1(CRL-2161), mouse MS1 (CRL-2279), mouse MS1 VEGF (CRL-2460), stem cells,etc. The phrase “capable of forming blood vessels” does not indicate aparticular cell-type, but simply that the cells in the population areable under appropriate conditions to form blood vessels. In somecircumstances, the population may be heterogeneous, comprising more thanone cell-type, only some which actually differentiate into bloodvessels, but others which are necessary to initiate, maintain, etc., theprocess of vessel formation.

The cell population can be contacted with the test agent in any mannerand under any conditions suitable for it to exert an effect on thecells, and to modulate the differentially-expressed gene or polypeptide.The means by which the test agent is delivered to the cells may dependupon the type of test agent, e.g., its chemical nature, and the natureof the cell population. Generally, a test agent must have access to thecell population, so it must be delivered in a form (or pro-form) thatthe population can experience physiologically, i.e., to put in contactwith the cells. For instance, if the intent is for the agent to enterthe cell, if necessary, it can be associated with any means thatfacilitate or enhance cell penetrance, e.g., associated with antibodiesor other reagents specific for cell-surface antigens, liposomes, lipids,chelating agents, targeting moieties, etc. Cells can also be treated,manipulated, etc., to enhance delivery, e.g., by electroporation,pressure variation, etc.

A purpose of administering or delivering the test agents to cellscapable of forming blood vessels is to determine whether they modulatethe gene or polypeptide. By the phrase “modulate,” it is meant that thegene or polypeptide affects the polypeptide or gene in some way.Modulation includes effects on transcription, RNA splicing, RNA editing,transcript stability and turnover, translation, polypeptide activity,and, in general, any process involved in the expression and productionof the gene and gene product. The modulatory activity can be in anydirection, and in any amount, including, up, down, enhance, increase,stimulate, activate, induce, turn on, turn off, decrease, block,inhibit, suppress, prevent, etc.

Any type of test agent can be used, comprising any material, such aschemical compounds, biomolecules, such as polypeptides (includingpolypeptide fragments and mimics), lipids, nucleic acids, carbohydrates,antibodies, small molecules, fusion proteins, etc. Test agents include,e.g., protamine (Taylor et al., Nature, 297:307, 1982), heparins,steroids, such as tetrahydrocortisol, which lack gluco- andmineral-corticoid activity (e.g., Folkman et al., Science, 221:719, 1983and U.S. Pat. Nos. 5,001,116 and 4,994,443), angiostatins (e.g., WO95/292420), triazines (e.g., U.S. Pat. No. 6,150,362), thrombospondins,endostatins, platelet factor 4, fumagillin-derivate AGH 1470,alpha-interferon, quinazolinones (e.g., U.S. Pat. No. 6,090,814),substituted dibenzothiophenes (e.g., U.S. Pat. No. 6,022,307),deoxytetracyclines, cytokines, chemokines, FGFs, etc.

Whether the test agent modulates a gene or polypeptide can be determinedby any suitable method. These methods include, detecting genetranscription, detecting mRNA, detecting polypeptide and activitythereof. The detection methods includes those mentioned herein, e.g.,PCR, RT-PCR, Northern blot, ELISA, Western, RIA, yeast two-hybrid system(e.g., for identifying natural and synthetic nucleic acids and theirproducts). In addition, further downstream targets can be used to assessthe effects of modulators, including, the presence or absence ofneoangiogenesis (e.g., using any of the mentioned test systems, such asCAM, BCE, in vivo Matrigel-type assays) as modulated by a test agent.

The present invention also relates to methods of regulating angiogenesisin a system comprising cells, comprising administering to the system aneffective amount of a modulator of a differentially-expressed gene orpolypeptide under conditions effective for the modulator to modulate thegene or polypeptide, whereby angiogenesis is regulated. A systemcomprising cells can be an in vivo system, such as a heart or limbpresent in a patient (e.g., angiogenic therapy to treat myocardialinfarction), isolated organs, tissues, or cells, in vitro assays systems(CAM, BCE, etc), animal models (e.g., in vivo, subcutaneous, chronicallyischemic lower limb in a rabbit model, cancer models), hosts in need oftreatment (e.g., hosts suffering from angiogenesis related diseases,such as cancer, ischemic syndromes, arterial obstructive disease, topromote collateral circulation, to promote vessel growth intobioengineered tissues, etc.

A modulator useful in such method are those mentioned already, e.g.,nucleic acid (such as an anti-sense to a gene to disrupt transcriptionor translation of the gene), antibodies (e.g., to inhibit a cell-surfaceprotein, such as an antibody specific-for the extracellular domain).Antibodies and other agents which target a polypeptide can be conjugatedto a cytotoxic or cytostatic agent, such as those mentioned already. Amodulator can also be a differentially-expressed gene, itself, e.g.,when it is desired to deliver the polypeptide to cells analogously togene therapy methods. A complete gene, or a coding sequence operablylinked to an expression control sequence (i.e., an expressible gene) canbe used to produce polypeptide in the target cells.

By the phrase “regulating angiogenesis,” it is meant that angiogenesisis effected in a desired way by the modulator. This includes,inhibiting, blocking, reducing, stimulating, inducing, etc., theformation of blood vessels. For instance, in cancer, where the growth ofnew blood vessels is undesirable, modulators of adifferentially-expressed can be used to inhibit their formation, therebytreating the cancer. Such inhibitory modulators include, e.g.,antibodies to the extracellular regions of a differentially-expressedpolypeptide, and, antisense RNA to inhibit translation of adifferentially-expressed mRNA into polypeptide (for guidance onadministering and designing anti-sense, see, e.g., U.S. Pat. Nos.6,153,595, 6,133,246, 6,117,847, 6,096,722, 6,087,343, 6,040,296,6,005,095, 5,998,383, 5,994,230, 5,891,725, 5,885,970, and 5,840,708).On the other hand, angiogenesis can be stimulated to treat ischemicsyndromes and arterial obstructive disease, to promote collateralcirculation, and to promote vessel growth into bio-engineered tissues,etc., by administering the a differentially-expressed gene orpolypeptide to a target cell population.

Markers

The polynucleotides of the present invention can be used with othermarkers, especially angiogenesis markers, to identity, detect, stage,diagnosis, determine, prognosticate, treat, etc., tissue, diseases andconditions, etc, of the vascular tissue. Markers can be polynucleotides,polypeptides, antibodies, ligands, specific binding partners, etc. Thetargets for such markers include, but are not limited genes andpolypeptides that are selective for angiogenesis and vascular tissues.

Therapeutics

Selective polynucleotides, polypeptides, and specific-binding partnersthereto, can be utilized in therapeutic applications, especially totreat diseases and conditions of vascular tissue, includingangiogenesis. Useful methods include, but are not limited to,immunotherapy (e.g., using specific-binding partners to polypeptides),vaccination (e.g., using a selective polypeptide or a naked DNA encodingsuch polypeptide), protein or polypeptide replacement therapy, genetherapy (e.g., germ-line correction, antisense), etc.

Various immunotherapeutic approaches can be used. For instance,unlabeled antibody that specifically recognizes a tissue-specificantigen can be used to stimulate the body to destroy or attack thecancer, to cause down-regulation, to produce complement-mediated lysis,to inhibit cell growth, etc., of target cells which display the antigen,e.g., analogously to how c-erbB-2 antibodies are used to treat breastcancer. In addition, antibody can be labeled or conjugated to enhanceits deleterious effect, e.g., with radionuclides and other energyemitting entitities, toxins, such as ricin, exotoxin A (ETA), anddiphtheria, cytotoxic or cytostatic agents, immunomodulators,chemotherapeutic agents, etc. See, e.g., U.S. Pat. No. 6,107,090.

An antibody or other specific-binding partner can be conjugated to asecond molecule, such as a cytotoxic agent, and used for targeting thesecond molecule to a tissue-antigen positive cell (Vitetta, E. S. etal., 1993, Immunotoxin therapy, in DeVita, Jr., V. T. et al., eds,Cancer: Principles and Practice of Oncology, 4th ed., J. B. LippincottCo., Philadelphia, 2624-2636). Examples of cytotoxic agents include, butare not limited to, antimetabolites, alkylating agents, anthracyclines,antibiotics, anti-mitotic agents, radioisotopes and chemotherapeuticagents. Further examples of cytotoxic agents include, but are notlimited to ricin, doxorubicin, daunorubicin, taxol, ethidium bromide,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,dihydroxy anthracin dione, actinomycin D, 1-dehydrotestosterone,diptheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, elongationfactor-2 and glucocorticoid. Techniques for conjugating therapeuticagents to antibodies are well.

In addition to immunotherapy, polynucleotides and polypeptides can beused as targets for non-immunotherapeutic applications, e.g., usingcompounds which interfere with function, expression (e.g., antisense asa therapeutic agent), assembly, etc. RNA interference can be used invitro and in vivo to silence a gene when its expression contributes to adisease (but also for other purposes, e.g., to identify the gene'sfunction to change a developmental pathway of a cell, etc.). See, e.g.,Sharp and Zamore, Science, 287:2431-2433, 2001; Grishok et al., Science,287:2494, 2001.

Delivery of therapeutic agents can be achieved according to anyeffective method, including, liposomes, viruses, plasmid vectors,bacterial delivery systems, orally, systemically, etc. Therapeuticagents of the present invention can be administered in any form by anyeffective route, including, e.g., oral, parenteral, enteral,intraperitoneal, topical, transdermal (e.g., using any standard patch),ophthalmic, nasally, local, non-oral, such as aerosal, inhalation,subcutaneous, intramuscular, buccal, sublingual, rectal, vaginal,intra-arterial, and intrathecal, etc. They can be administered alone, orin combination with any ingredient(s), active or inactive.

In addition to therapeutics, per se, the present invention also relatesto methods of treating a diseases and conditions of the vasculartissues, comprising, e.g., administering to a subject in need thereof atherapeutic agent which is effective for regulating a gene orpolypeptide and/or which is effective in treating said disease orcondition. The term “treating” is used conventionally, e.g., themanagement or care of a subject for the purpose of combating,alleviating, reducing, relieving, improving the condition of, etc., of adisease or disorder. Diseases or disorders which can be treated inaccordance with the present invention include, but are not limited toinflammatory diseases, such as rheumatoid arthritis, osteoarthritis,asthma, pulmonary fibrosis, age-related macular degeneration (ARMD),diabetic retinopathy, macular degeneration, and retinopathy ofprematurity (ROP), endometriosis, cancer, Coats' disease, peripheralretinal neovascularization, neovascular glaucoma, psoriasis, retrolentalfibroplasias, angiofibroma, inflammation, and any of the disorderslisted in Table 2, etc.

By the phrase “altered expression,” it is meant that the disease isassociated with a mutation in the gene, or any modification to the gene(or corresponding product) which affects its normal function. Thus,expression of a gene refers to, e.g., transcription, translation,splicing, stability of the mRNA or protein product, activity of the geneproduct, differential expression, etc.

Any agent which “treats” the disease can be used. Such an agent can beone which regulates its expression. Expression refers to the same actsalready mentioned, e.g. transcription, translation, splicing, stabilityof the mRNA or protein product, activity of the gene product,differential expression, etc. For instance, if the condition was aresult of a complete deficiency of the gene product, administration ofgene product to a patient would be said to treat the disease andregulate the gene's expression. Many other possible situations arepossible, e.g., where the gene is aberrantly expressed, and thetherapeutic agent regulates the aberrant expression by restoring itsnormal expression pattern.

Antisense

Antisense polynucleotide (e.g., RNA) can also be prepared from apolynucleotide according to the present invention, preferably ananti-sense to a sequence of SEQ ID NO 1-58. Antisense polynucleotide canbe used in various ways, such as to regulate or modulate expression ofthe polypeptides they encode, e.g., inhibit their expression, for insitu hybridization, for therapeutic purposes, for making targetedmutations (in vivo, triplex, etc.) etc. For guidance on administeringand designing anti-sense, see, e.g., U.S. Pat. Nos. 6,200,960,6,200,807, 6,197,584, 6,190,869, 6,190,661, 6,187,587, 6,168,950,6,153,595, 6,150,162, 6,133,246, 6,117,847, 6,096,722, 6,087,343,6,040,296, 6,005,095, 5,998,383, 5,994,230, 5,891,725, 5,885,970, and5,840,708. An antisense polynucleotides can be operably linked to anexpression control sequence. A total length of about 35 bp can be usedin cell culture with cationic liposomes to facilitate cellular uptake,but for in vivo use, preferably shorter oligonucleotides areadministered, e.g. 25 nucleotides.

Antisense polynucleotides can comprise modified, nonnaturally-occurringnucleotides and linkages between the nucleotides (e.g., modification ofthe phosphate-sugar backbone; methyl phosphonate, phosphorothioate, orphosphorodithioate linkages; and 2′-O-methyl ribose sugar units), e.g.,to enhance in vivo or in vitro stability, to confer nuclease resistance,to modulate uptake, to modulate cellular distribution andcompartmentalization, etc. Any effective nucleotide or modification canbe used, including those already mentioned, as known in the art, etc.,e.g., disclosed in U.S. Pat. Nos. 6,133,438; 6,127,533; 6,124,445;6,121,437; 5,218,103 (e.g., nucleoside thiophosphoramidites); 4,973,679;Sproat et al., “2′-O-Methyloligoribonucleotides: synthesis andapplications,” Oligonucleotides and Analogs A Practical Approach,Eckstein (ed.), IRL Press, Oxford, 1991, 49-86; Iribarren et al.,“2′O-Alkyl Oligoribonucleotides as Antisense Probes,” Proc. Natl. Acad.Sci. USA, 1990, 87, 7747-7751; Cotton et al., “2′-O-methyl, 2′-O-ethyloligoribonucleotides and phosphorothioate oligodeoxyribonucleotides asinhibitors of the in vitro U7 snRNP-dependent mRNA processing event,”Nucl. Acids Res., 1991, 19, 2629-2635.

Arrays

The present invention also relates to an ordered array of polynucleotideprobes and specific-binding partners (e.g., antibodies) for detectingthe expression of a gene or polypeptide of the present invention in asample, comprising, one or more polynucleotide probes or specificbinding partners associated with a solid support, wherein each probe isspecific for said gene or polypeptide, The probes can comprise anucleotide sequence of SEQ ID NO 1-58 which is specific for said gene, anucleotide sequence having sequence identity to SEQ ID NO 1-58 which isspecific for said gene or polynucleotide, or complements thereto, or aspecific-binding partner which is specific for said genes.

The phrase “ordered array” indicates that the probes are arranged in anidentifiable or position-addressable pattern, e.g., such as the arraysdisclosed in U.S. Pat. Nos. 6,156,501, 6,077,673, 6,054,270, 5,723,320,5,700,637, WO09919711, WO00023803. The probes are associated with thesolid support in any effective way. For instance, the probes can bebound to the solid support, either by polymerizing the probes on thesubstrate, or by attaching a probe to the substrate. Association can be,covalent, electrostatic, noncovalent, hydrophobic, hydrophilic,noncovalent, coordination, adsorbed, absorbed, polar, etc. When fibersor hollow filaments are utilized for the array, the probes can fill thehollow orifice, be absorbed into the solid filament, be attached to thesurface of the orifice, etc. Probes can be of any effective size,sequence identity, composition, etc., as already discussed.

Ordered arrays can further comprise polynucleotide probes orspecific-binding partners which are specific for other genes, includinggenes specific for angiogenesis or vascular tissues.

Transgenic Animals

The present invention also relates to transgenic animals comprising oneor genes of the present invention. Such genes, as discussed in moredetail below, include, but are not limited to, functionally-disruptedgenes, mutated genes, ectopically or selectively-expressed genes,inducible or regulatable genes, etc. These transgenic animals can beproduced according to any suitable technique or method, includinghomologous recombination, mutagenesis (e.g., ENU, Rathkolb et al., Exp.Physiol., 85(6):635-644, 2000), and the tetracycline-regulated geneexpression system (e.g., U.S. Pat. No. 6,242,667). The term “gene” asused herein includes any part of a gene, i.e., regulatory sequences,promoters, enhancers, exons, introns, coding sequences, etc. The nucleicacid present in the construct or transgene can be naturally-occurringwild-type, polymorphic, or mutated.

Along these lines, polynucleotides of the present invention can be usedto create transgenic animals, e.g. a non-human animal, comprising atleast one cell whose genome comprises a functional disruption of a geneof the present invention, e.g., represented by the genes set forth inTables 1-3. By the phrases “functional disruption” or “functionallydisrupted,” it is meant that the gene does not express abiologically-active product. It can be substantially deficient in atleast one functional activity coded for by the gene. Expression of apolypeptide can be substantially absent, i.e., essentially undetectableamounts are made. However, polypeptide can also be made, but which isdeficient in activity, e.g., where only an amino-terminal portion of thegene product is produced. Such an animal can show aberrant or defectiveangiogenesis (e.g., angiogenesis is increased or decreased, such asexcessive or extraneous angiogenesis, or insufficient angiogenesis orvascularization), leading to a host of effects on different organsystems.

The transgenic animal can comprise one or more cells. When substantiallyall its cells contain the engineered gene, it can be referred to as atransgenic animal “whose genome comprises” the engineered gene. Thisindicates that the endogenous gene loci of the animal has been modifiedand substantially all cells contain such modification.

Functional disruption of the gene can be accomplished in any effectiveway, including, e.g., introduction of a stop codon into any part of thecoding sequence such that the resulting polypeptide is biologicallyinactive (e.g., because it lacks a catalytic domain, a ligand bindingdomain, etc.), introduction of a mutation into a promoter or otherregulatory sequence that is effective to turn it off, or reducetranscription of the gene, insertion of an exogenous sequence into thegene which inactivates it (e.g., which disrupts the production of abiologically-active polypeptide or which disrupts the promoter or othertranscriptional machinery), deletion of sequences from the gene, etc.Insertions can be made in the novel parts of the genes as shown in theattached figures. Examples of transgenic animals having functionallydisrupted genes are well known, e.g., as described in U.S. Pat. Nos.6,239,326, 6,225,525, 6,207,878, 6,194,633, 6,187,992, 6,180,849,6,177,610, 6,100,445, 6,087,555, 6,080,910, 6,069,297, 6,060,642,6,028,244, 6,013,858, 5,981,830, 5,866,760, 5,859,314, 5,850,004,5,817,912, 5,789,654, 5,777,195, and 5,569,824. A transgenic animalwhich comprises the functional disruption can also be referred to as a“knock-out” animal, since the biological activity of said gene has been“knocked-out.” Knock-outs can be homozygous or heterozygous.

For creating functional disrupted genes, and other gene mutations,homologous recombination technology is of special interest since itallows specific regions of the genome to be targeted. Using homologousrecombination methods, genes can be specifically-inactivated, specificmutations can be introduced, and exogenous sequences can be introducedat specific sites. These methods are well known in the art, e.g., asdescribed in the patents above. See, also, Robertson, Biol. Reproduc.,44(2):238-245, 1991. Generally, the genetic engineering is performed inan embryonic stem (ES) cell, or other pluripotent cell line (e.g., adultstem cells, EG cells), and that genetically-modified cell (or nucleus)is used to create a whole organism. Nuclear transfer can be used incombination with homologous recombination technologies.

For example, the endogenous locus can be disrupted in mouse ES cellsusing a positive-negative selection method (e.g., Mansour et al.,Nature, 336:348-352, 1988). In this method, a targeting vector can beconstructed which comprises a part of the gene to be targeted. Aselectable marker, such as neomycin resistance genes, can be insertedinto a an exon present in the targeting vector, disrupting it. When thevector recombines with the ES cell genome, it disrupts the function ofthe gene. The presence in the cell of the vector can be determined byexpression of neomycin resistance. See, e.g., U.S. Pat. No. 6,239,326.Cells having at least one functionally disrupted gene can be used tomake chimeric and germline animals, e.g., animals having somatic and/orgerm cells comprising the engineered gene. Homozygous knock-out animalscan be obtained from breeding heterozygous knock-out animals. See, e.g.,U.S. Pat. No. 6,225,525.

A transgenic animal, or animal cell, lacking one or more functionalactiviteis can be used to decipher angiogenesis, or any of the utilitiesmentioned in any issued U.S. patent on transgenic animals, including,U.S. Pat. Nos. 6,239,326, 6,225,525, 6,207,878, 6,194,633, 6,187,992,6,180,849, 6,177,610, 6,100,445, 6,087,555, 6,080,910, 6,069,297,6,060,642, 6,028,244, 6,013,858, 5,981,830, 5,866,760, 5,859,314,5,850,004, 5,817,912, 5,789,654, 5,777,195, and 5,569,824. For instance,deficient animal cells can be utilized to study angiogenesis. Byknocking-out the genes which are involved in angiogenesis, e.g., one ata time, the physiological pathways can be dissected out and identified.

The present invention also relates to non-human, transgenic animal whosegenome comprises a recombinant nucleic acid of the present invention(e.g., SEQ ID NOS 1-58) operatively linked to an expression controlsequence effective to express said coding sequence, e.g., in vascularand endothelial tissues. Such a transgenic animal can also be referredto as a “knock-in” animal since an exogenous gene has been introduced,stably, into its genome. For instance, the endogenous locus can beknocked-out, and a polynucleotide of the present invention, e.g., SEQ IDNOS 1-58, can be inserted.

A recombinant nucleic acid refers to a gene which has been introducedinto a target host cell and optionally modified, such as cells derivedfrom animals, plants, bacteria, yeast, etc. A recombinant nucleic acidincludes completely synthetic nucleic acid sequences, semi-syntheticnucleic acid sequences, sequences derived from natural sources, andchimeras thereof. “Operable linkage” has the meaning used through thespecification, i.e., placed in a functional relationship with anothernucleic acid. When a gene is operably linked to an expression controlsequence, as explained above, it indicates that the gene (e.g., codingsequence) is joined to the expression control sequence (e.g., promoter)in such a way that facilitates transcription and translation of thecoding sequence. As described above, the phrase “genome” indicates thatthe genome of the cell has been modified. In this case, the recombinantnucleic acid has been stably integrated into the genome of the animal.The nucleic acid in operable linkage with the expression controlsequence can also be referred to as a construct or transgene.

Any expression control sequence can be used depending on the purpose.For instance, if selective expression is desired, then expressioncontrol sequences which limit its expression can be selected. Theseinclude, e.g., tissue or cell-specific promoters, introns, enhancers,etc. For various methods of cell and tissue-specific expression, see,e.g., U.S. Pat. Nos. 6,215,040, 6,210,736, and 6,153,427. These alsoinclude the endogenous promoter, i.e., the coding sequence can beoperably linked to its own promoter. Inducible and regulatable promoterscan also be utilized.

The present invention also relates to a transgenic animal which containsa functionally disrupted and a transgene stably integrated into theanimals genome. Such an animal can be constructed using combinations anyof the above- and below-mentioned methods. Such animals have any of theaforementioned uses, including permitting the knock-out of the normalgene and its replacement with a mutated gene. Such a transgene can beintegrated at the endogenous gene locus so that the functionaldisruption and “knock-in” are carried out in the same step.

In addition to the methods mentioned above, transgenic animals can beprepared according to known methods, including, e.g., by pronuclearinjection of recombinant genes into pronuclei of 1-cell embryos,incorporating an artificial yeast chromosome into embryonic stem cells,gene targeting methods, embryonic stem cell methodology, cloningmethods, nuclear transfer methods. See, also, e.g., U.S. Pat. Nos.4,736,866; 4,873,191; 4,873,316; 5,082,779; 5,304,489; 5,174,986;5,175,384; 5,175,385; 5,221,778; Gordon et al., Proc. Natl. Acad. Sci.,77:7380-7384, 1980; Palmiter et al., Cell, 41:343-345, 1985; Palmiter etal., Ann. Rev. Genet., 20:465-499, 1986; Askew et al., Mol. Cell. Bio.,13:4115-4124, 1993; Games et al. Nature, 373:523-527, 1995; Valanciusand Smithies, Mol. Cell. Bio., 11:1402-1408, 1991; Stacey et al., Mol.Cell. Bio., 14:1009-1016, 1994; Hasty et al., Nature, 350:243-246, 1995;Rubinstein et al., Nucl. Acid Res., 21:2613-2617,1993; Cibelli et al.,Science, 280:1256-1258, 1998. For guidance on recombinase excisionsystems, see, e.g., U.S. Pat. Nos. 5,626,159, 5,527,695, and 5,434,066.See also, Orban, P. C., et al., “Tissue- and Site-Specific DNARecombination in Transgenic Mice,” Proc. Natl. Acad. Sci. USA,89:6861-6865 (1992); O'Gorman, S., et al., “Recombinase-Mediated GeneActivation and Site-Specific Integration in Mammalian Cells,” Science,251:1351-1355 (1991); Sauer, B., et al., “Cre-stimulated recombinationat loxP-Containing DNA sequences placed into the mammalian genome,”Polynucleotides Research, 17(1): 147-161 (1989); Gagneten, S. et al.(1997) Nucl. Acids Res. 25:3326-3331; Xiao and Weaver (1997) Nucl. AcidsRes. 25:2985-2991; Agah, R. et al. (1997) J. Clin. Invest. 100:169-179;Barlow, C. et al. (1997) Nucl. Acids Res. 25:2543-2545; Araki, K. et al.(1997) Nucl. Acids Res. 25:868-872; Mortensen, R. N. et al. (1992) Mol.Cell. Biol. 12:2391-2395 (G418 escalation method); Lakhlani, P. P. etal. (1997) Proc. Natl. Acad. Sci. USA 94:9950-9955 (“hit and run”);Westphal and Leder (1997) Curr. Biol. 7:530-533 (transposon-generated“knock-out” and “knock-in”); Templeton, N. S. et al. (1997) Gene Ther.4:700-709 (methods for efficient gene targeting, allowing for a highfrequency of homologous recombination events, e.g., without selectablemarkers); PCT International Publication WO 93/22443(functionally-disrupted).

A polynucleotide according to the present invention can be introducedinto any non-human animal, including a non-human mammal, mouse (Hogan etal., Manipulating the Mouse Embryo: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1986), pig (Hammer et al.,Nature, 315:343-345, 1985), sheep (Hammer et al., Nature, 315:343-345,1985), cattle, rat, or primate. See also, e.g., Church, 1987, Trends inBiotech. 5:13-19; Clark et al., Trends in Biotech. 5:20-24, 1987); andDePamphilis et al., BioTechniques, 6:662-680, 1988. Transgenic animalscan be produced by the methods described in U.S. Pat. No. 5,994,618, andutilized for any of the utilities described therein.

Database

The present invention also relates to electronic forms ofpolynucleotides, polypeptides, etc., of the present invention, includingcomputer-readable medium (e.g., magnetic, optical, etc., stored in anysuitable format, such as flat files or hierarchical files) whichcomprise such sequences, or fragments thereof, e-commerce-related means,etc. Along these lines, the present invention relates to methods ofretrieving gene sequences from a computer-readable medium, comprising,one or more of the following steps in any effective order, e.g.,selecting a cell or gene expression profile, e.g., a profile thatspecifies that said gene is expressed in blood vessels, and retrievingsaid gene sequence, where the gene sequence is represented by SEQ ID NOS1-58.

A “gene expression profile” means the list of tissues, cells, etc., inwhich a defined gene is expressed (i.e, transcribed and/or translated).A “cell expression profile” means the genes which are expressed in theparticular cell type. The profile can be a list of the tissues in whichthe gene is expressed, but can include additional information as well,including level of expression (e.g., a quantity as compared ornormalized to a control gene), and information on temporal (e.g., atwhat point in the cell-cycle or developmental program) and spatialexpression. By the phrase “selecting a gene or cell expression profile,”it is meant that a user decides what type of gene or cell expressionpattern he is interested in retrieving, e.g., he may require that thegene is differentially expressed in a tissue, or he may require that thegene is not expressed in heart, but must be expressed in cells capableof forming blood vessels. Any pattern of expression preferences may beselected. The selecting can be performed by any effective method. Ingeneral, “selecting” refers to the process in which a user forms a querythat is used to search a database of gene expression profiles. The stepof retrieving involves searching for results in a database thatcorrespond to the query set forth in the selecting step. Any suitablealgorithm can be utilized to perform the search query, includingalgorithms that look for matches, or that perform optimization betweenquery and data. The database is information that has been stored in anappropriate storage medium, having a suitable computer-readable format.Once results are retrieved, they can be displayed in any suitableformat, such as HTML.

For instance, the user may be interested in identifying genes that areexpressed in a vascular tissue. He may not care whether small amounts ofexpression occur in other tissues, as long as such genes are notexpressed in peripheral blood lymphocytes. A query is formed by the userto retrieve the set of genes from the database having the desired geneor cell expression profile. Once the query is inputted into the system,a search algorithm is used to interrogate the database, and retrieveresults.

The present invention also relates to methods of selecting a geneexpressed in vascular tissue (e.g., during angiogenesis) from a databasecomprising polynucleotide sequences, comprising displaying, in acomputer-readable medium, a polynucleotide sequence or polypeptidesequence for SEQ ID NOS 1-58, or complements to the polynucleotidesequence, wherein said displayed sequences have been retrieved from saiddatabase upon selection by a user. The phrase “upon selection by a user”indicates that a user of the database has specified or directed a searchor other retrieval feature that results in the retrieval and display ofthe target sequences. For example, the user could ask the database todisplay polynucleotides or polypeptides expressed during angiogenesis byinputting an appropriate inquiry. The user could also input sequenceinformation, and request the display of any sequences in the databasethat match the inputted sequence information. One or more sequences canbe displayed at a time in response to any user inquiry.

Advertising, Licensing, etc., Methods

The present invention also relates to methods of advertising, licensing,selling, purchasing, brokering, etc., genes, polynucleotides,specific-binding partners, antibodies, etc., of the present invention.Methods can comprises, e.g., displaying a gene, polypeptide, or antibodyspecific thereto in a printed or computer-readable medium (e.g., on theWeb or Internet), accepting an offer to purchase said gene, polypeptide,or antibody.

Other

A polynucleotide, probe, polypeptide, antibody, specific-bindingpartner, etc., according to the present invention can be isolated. Theterm “isolated” means that the material is in a form in which it is notfound in its original environment or in nature, e.g., more concentrated,more purified, separated from component, etc. An isolated polynucleotideincludes, e.g., a polynucleotide having the sequenced separated from thechromosomal DNA found in a living animal, e.g., as the complete gene, atranscript, or a cDNA. This polynucleotide can be part of a vector orinserted into a chromosome (by specific gene-targeting or by randomintegration at a position other than its normal position) and still beisolated in that it is not in a form that is found in its naturalenvironment. A polynucleotide, polypeptide, etc., of the presentinvention can also be substantially purified. By substantially purified,it is meant that polynucleotide or polypeptide is separated and isessentially free from other polynucleotides or polypeptides, i.e., thepolynucleotide or polypeptide is the primary and active constituent. Apolynucleotide can also be a recombinant molecule. By “recombinant,” itis meant that the polynucleotide is an arrangement or form which doesnot occur in nature. For instance, a recombinant molecule comprising apromoter sequence would not encompass the naturally-occurring gene, butwould include the promoter operably linked to a coding sequence notassociated with it in nature, e.g., a reporter gene, or a truncation ofthe normal coding sequence.

The term “marker” is used herein to indicate a means for detecting orlabeling a target. A marker can be a polynucleotide (usually referred toas a “probe”), polypeptide (e.g., an antibody conjugated to a detectablelabel), PNA, or any effective material.

The topic headings set forth above are meant as guidance where certaininformation can be found in the application, but are not intended to bethe only source in the application where information on such topic canbe found. Reference materials For other aspects of the polynucleotides,reference is made to standard textbooks of molecular biology. See, e.g.,Hames et al., Polynucleotide Hybridization, IL Press, 1985; Davis etal., Basic Methods in Molecular Biology, Elsevir Sciences Publishing,Inc., New York, 1986; Sambrook et al., Molecular Cloning, CSH Press,1989; Howe, Gene Cloning and Manipulation, Cambridge University Press,1995; Ausubel et al., Current Protocols in Molecular Biology, John Wiley& Sons, Inc., 1994-1998.

The preceding description, utilize the present invention to its fullestextent. The preceding preferred specific embodiments are, therefore, tobe construed as merely illustrative, and not limiting the remainder ofthe disclosure in any way whatsoever. The entire disclosure of allapplications, patents and publications, cited above and in the figuresare hereby incorporated by reference in their entirety. TABLE 1 Name RefSeq Domain Domain description Begin End ANH0009 XM_087061: similar torrm RNA recognition motif 37 198 heterogeneous nuclear ribonucleoproteinA3 ANH0024A X68560: SPR2 zf-C2H2 Zinc finger, C2H2 type 621 703transcription factor ANH0024A Autotransporter Autotransporterbeta-domain 197 463 ANH0024B zf-C2H2 Zinc finger, C2H2 type 580 662ANH0024B Autotransporter Autotransporter beta-domain 156 422 ANH0024Czf-C2H2 Zinc finger, C2H2 type 336 418 ANH0024D zf-C2H2 Zinc finger,C2H2 type 595 677 ANH0024D Autotransporter Autotransporter beta-domain171 437 ANH0039 XM_045848: calumenin efhand EF hand 72 297 ANH0068XM_055371: uridine No domain found 5′monophosphate hydrolase 1 (UMPH1)ANH0114 XM_076374: similar to Kelch Kelch motif (4) 348 600Kelch-related protein 1 ANH0114 BTB BTB/POZ domain 23 128 ANH0144AXM_098238; AL133047: SH3 SH3 domain 778 1067 similar to SH3 domainprotein D19 ANH0144A Tymo_45 kd_70 kd Tymovirus 45/70 Kd protein 261 624ANH0144B SH3 SH3 domain 863 993 ANH0144B Atrophin-1 Atrophin-1 family 10787 ANH0144C SH3 SH3 domain 781 1070 ANH0144C Tymo_45 kd_70 kd Tymovirus45/70 Kd protein 264 627 ANH0241 XM_086643 PHD PHD-finger 90 133 ANH0241bromodomain Bromodomain 154 240 ANH0241 zf-MYND MYND finger 1028 1062ANH0241 PWWP PWWP domain 274 345 ANH0241 Parathyroid Parathyroid hormonefamily 517 638 ANH0241 Granin Granin (chromogranin or 410 989secretogranin ANH0241 zf-B_box B-box zinc finger 1023 1067 ANH0245NM_032847; AK027731 No domain found ANH0296 AK000913; NM_004713Ribosomal_L22 Ribosomal protein L22p/L17e 328 412 ANH0296 Caulimo_VICaulimovirus viroplasmin 386 795 ANH0296 Peripla_BP_2 Periplasmicbinding protein 223 465 ANH0296 SART-1 SART-1 family 385 930 ANH0423XM_053487: FGD1 RhoGEF Guanine exchange factor for Rho- 161 340 familymember like GTPases PH Pleckstrin homology domain 371 471 FYVE Proteinpresent in Fab1, YOTB, 524 589 Vac1, and EEA1 PH Pleckstrin homologydomain 605 705 ANH0459B NM_000366: Tropomyosin Tropomyosin 48 284tropomyosin 1 (TPM1) ANH0459C Tropomyosin Tropomyosin 12 244 ANH0459Cspectrin Spectrin repeat 145 244 ANH0459D Tropomyosin Tropomyosin 1 158ANH0769 XM_038985; AL133087 ank Ankyrin repeat 7 991 ANH0769 AvirulenceXanthomonas avirulence protein, 205 664 Avr/P ANH0658 NM_014882 RhoGAPRhoGAP domain 177 331 ANH0658 PH PH domain 47 151 ANH0658 Peptidase_S9_NProlyl oligopeptidase, N-terminal 16 355 bet ANH0668 XM_015539 TMTransmembrane domain 65 87 TM Transmembrane domain 97 119 ANH0757XM_087631 bZIP bZIP transcription factor 519 583 ANH0687A XM_048092 WH1WH1 domain 1 101 ANH0687A Ran_BP1 RanBP1 domain 5 101 ANH0687B WH1 WH1domain 1 101 ANH0687B Ran_BP1 RanBP1 domain 5 103 ANH0687B Armadillo_segArmadillo/beta-catenin-like repeat 352 392 ANH0693 NM_052877 No domainfound ANH0095 AK023027 TM Transmembrane 40 62 ANH0122A RRM RNArecognition motif 88 160 Coiled coil 271 352 Coiled coil 385 554 PWIPWI, domain in splicing factor 763 836 ANH0122B RRM RNA recognitionmotif 88 160 Coiled coil 271 490 PWI PWI, domain in splicing factor 699772 ANH0316 NM_005807; Somatomedin_B Somatomedin B domain 25 68XM_001738: proteoglycan 4 ANH0316 hemopexin Hemopexin 1067 1157 ANH0316GASA Gibberellin regulated protein 4 72 ANH0316 wap WAP-type (WheyAcidic Protein) 29 66

TABLE 2 Clone ID Locus Associated Diseases ANH0009 2q31.2 Erythermalgia,Familial Primary; Cancer; Primary Pulmonary Hypertension locus ANH0024A2q37 Early pregnancy loss and stillborns; ANH0024B Autism; ANH0024CCancer, including oral squamous cell carcinomas; ANH0024D Brachydactyly,Type E (Bde); Systemic Lupus Erythematosus, Susceptibility To, 2(Sleb2); Brachydactyly-Mental Retardation Syndrome; Holoprosencephaly 6ANH0039 7q36 Cancer; Holoprosencephaly (HPE) Polydactyly, Preaxial Ii(Ppd2); Acropectoral Syndrome ANH0068 7p15.3 Deafness, AutosomalDominant Nonsyndromic Sensorineural 5 (Dfna5); Retinitis Pigmentosa 9(Rp9); *Stroke And Cerebral Cavernous Malformations 2 (Ccm2) ANH01143p21 Cancer; Modifier of Hirschsprung disease (HSCR), Aicardi-GoutieresSyndrome 1 (Ags1); Spinocerebellar Ataxia 7 (Sca7); Larsen Syndrome,Autosomal Dominant (Lrs1); *Vasculopathy, Retinal, With CerebralLeukodystrophy ANH0144A 4q31 Deafness, Autosomal Recessive 26 (Dfnb26);ANH0144B Cancer; ANH0144C Schizophrenia susceptibility locus ANH024120q13.3 Complex Obesity Trait ANH0245 8q24.13 Spastic Paraplegia 8,Autosomal Dominant (Spg8); Childhood Absence Epilepsy (Cae); Epilepsy,Myoclonic, Benign Adult Familial; *Epidermolysis Bullosa Simplex, OgnaType; Macular Dystrophy, Atypical Vitelliform (Vmd1); Tibial HemimeliaANH0296 14q21 Deafness, Autosomal Dominant Nonsyndromic Sensorineural 23(DFNA23) ANH0423 9q22 Cataract, Autosomal Recessive, Early-Onset,Pulverulent; Hemophagocytic Lymphohistiocytosis, Familial, 1;Amyotrophic Lateral Sclerosis With Frontotemporal Dementia;Nephronophthisis 2 (NPHP2) ANH0459B 15q22.1 Type 3 Familial HypertrophicCardiomyopathy (mutations in ANH0459C tropomyosin gene associated withdisease, e.g., Thierfelder et ANH0459D al., Cell, 77: 701-712, 1994)NH0769 2q33.1 Paroxysmal Nonkinesigenic Dyskinesia (Pnkd); Ichthyosis,Lamellar, 2 (Li2) ANH0658 2p13.1 Spastic Paraplegia 17 ANH0668 11q12Hereditary Spastic Paraplegia; Psoriasis; Breast Carcinoma and othercancers; Osteoporosis-pseudoglioma syndrome ANH0757 5q35.2 Congenitaldevelopment disorder (Zhu et al., Am. J. Med. Genet., 98: 317-9, 2001)ANH0687A 2p16.1 Cancer; ANH0687B Carney Complex ANH0693 1p34.1 Ptosis,Hereditary Congenital 1 (PTOS1) ANH0095 14q32.33 Immunoglobulin HeavyChain Regulator, Included (IGHR) ANH122 14q24.3 Leber congenitalamaurosis type III; Familial arrhythmogenic right ventriculardysplasia-1 (ARVD1) ANH0316 1q25-q31 Febrile Convulsions And TemporalLobe Epilepsy

TABLE 3 Protein- Length Clone ID (amino acids) SEQ ID NO ExpressionANH0009 358 1, 2 D24 L ANH0024A 781 3, 4 U8S L ANH0024B 740 5, 6ANH0024C 496 7, 8 ANH0024D 755 9, 10 ANH0039 315 11, 12 D8S L ANH0068297 13, 14 U1S L ANH0114 621 15, 16 U8T H ANH0144A 1070 17, 18 D8S LANH0144B 1023 19, 20 ANH0144C 1073 21, 22 ANH0241 1214 23, 24 D1T LANH0245 359 25, 26 D1T L ANH0296 1082 27, 28 U1S H ANH0423 725 29, 30U1T L ANH0459B 284 31, 32 D1S L ANH0459C 245 33, 34 ANH0459D 158 35, 36ANH0769 994 37, 38 U1S L ANH0658 645 39, 40 U1T L ANH0668 186 41, 42 D1TL ANH0757 639 43, 44 U1S H ANH0687A 641 45, 46 D1T L ANH0687B 817 47, 48ANH0693 268 49, 50 D8S L ANH0095 65 51, 52 U1S H ANH122A 843 53, 54 U1SL ANH122B 779 55, 56 ANH0316 1320 57, 58 U8T L

1. An isolated polynucleotide which codes without interruption for a human differentially-regulated human angiogenesis polypeptide having an amino acid sequence as set forth in SEQ ID NOS 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58, or a complement thereto.
 2. An isolated differentially-regulated human angiogenesis polynucleotide having a polynucleotide sequence as set forth in SEQ ID NOS 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57, or a complement thereto, and which is differentially-regulated in angiogenesis.
 3. An isolated polynucleotide comprising, a polynucleotide sequence coding without interruption for a differentially-regulated human angiogenesis polypeptide, or a complement thereto, said polypeptide having 90% or more amino acid sequence identity along its entire length to the polypeptide sequence as set forth in SEQ ID NOS 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or
 58. 4. An isolated polynucleotide comprising, a polynucleotide sequence coding without interruption for a differentially-regulated human angiogenesis polypeptide and having 90% or more nucleotide sequence identity along its entire length to a polynucleotide sequence as set forth in SEQ ID NOS 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57, or a complement thereto.
 5. An isolated polynucleotide which is specific for a differentially-regulated human angiogenesis gene of claim 1 and having a polynucleotide sequence selected from a polynucleotide sequence as set forth in SEQ ID NOS 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57, or a complement thereto.
 6. An isolated polynucleotide of claim 5, wherein said fragment is effective in a polymerase chain reaction.
 7. An isolated differentially-regulated human angiogenesis polypeptide of claim
 1. 8. An isolated differentially-regulated human angiogenesis polypeptide of claim
 2. 9. An isolated differentially-regulated human angiogenesis polypeptide of claim
 3. 10. An isolated differentially-regulated human angiogenesis polypeptide of claim
 4. 11. A method of detecting a nucleic acid coding for a differentially-regulated human angiogenesis gene, comprising, contacting a sample comprising nucleic acid with a polynucleotide probe specific for a differentially-regulated human angiogenesis gene of claim 1 under conditions effective for said probe to hybridize specifically with said gene, and detecting hybridization between said probe and said nucleic acid.
 12. A method of claim 11, wherein said detecting is performed by: Northern blot analysis, polymerase chain reaction (PCR), reverse transcriptase PCR, RACE PCR, or in situ hybridization.
 13. A method of detecting a nucleic acid coding for a differentially-regulated human angiogenesis gene, comprising, contacting a sample comprising nucleic acid with a polynucleotide probe specific for a differentially-regulated human angiogenesis gene of claim 3 under conditions effective for said probe to hybridize specifically with said gene, and detecting hybridization between said probe and said nucleic acid.
 14. A method of claim 13, wherein said detecting is performed by: Northern blot analysis, polymerase chain reaction (PCR), reverse transcriptase PCR, RACE PCR, or in situ hybridization.
 15. A method of treating a vascular disease or a disease associated with vascularization, comprising: administering to a subject in need thereof a therapeutic agent which is effective for regulating expression of a differentially-regulated angiogenesis polynucleotide of claim 1, or a polypeptide encoded thereby.
 16. A method of claim 15, wherein said agent is an antibody specific for said polypeptide.
 17. A method for identifying an agent that modulates the expression of a differentially-regulated angiogenesis polynucleotide, or polypeptide encoded thereby, in cells capable of forming blood vessels, comprising: contacting said cells with a test agent under conditions effective for said test agent to modulate the expression of a differentially-regulated angiogenesis polynucleotide of claim 1, or polypeptide encoded thereby, in said cells, and determining whether said test agent modulates said polynucleotide or polypeptide.
 18. A method of determining the angiogenic index of a sample comprising cells, comprising: assessing, in said sample, the expression level of polynucleotide of claim 1, or a polypeptide encoded thereof, whereby said levels are indicative of the angiogenic index.
 19. A method of claim 18, wherein the angiogenic index is assessed by polymerase chain reaction using polynucleotide primers specific for said polynucleotide.
 20. A method of claim 18, wherein the angiogenic index is assessed by detecting polypeptides coded for by said polynucleotides using specific antibodies.
 21. A method of regulating angiogenesis in a system comprising cells capable of forming blood vessels, comprising: administering to said system an effective amount of a modulator of a polynucleotide of claim 1, or a polypeptide coded thereby, under conditions effective for the modulator to modulate said polypeptide, whereby angiogenesis is regulated.
 22. A method of claim 21, wherein the modulator is an antibody specific-for said polypeptide.
 23. A method of claim 21, wherein the antibody is conjugated to a cytotoxic or cytostatic agent.
 24. A method of claim 21, wherein regulating angiogenesis is inhibiting angiogenesis or stimulating angiogenesis.
 25. A method of claim 21, wherein the system is a patent having a cancer, coronary artery disease, myocardial ischemia, or coronary arteriosclerosis.
 26. A method of detecting polymorphisms in a differentially-regulated human angiogenesis gene, comprising, comparing the structure of: genomic DNA comprising all or part of a differentially-regulated human angiogenesis gene, mRNA comprising all or part of a differentially-regulated human angiogenesis gene, cDNA comprising all or part of a differentially-regulated human angiogenesis gene, or a polypeptide comprising all or part of a differentially-regulated human angiogenesis gene, with the complete structure of a differentially-regulated human angiogenesis gene of claim
 2. 27. A method of claim 26, wherein said polymorphism is a nucleotide deletion, substitution, inversion, or transposition.
 28. A mammalian cell whose genome comprises a functional disruption of a differentially-regulated human angiogenesis polynucleotide of claim 1, or a mammalian homolog thereof.
 29. A non-human, transgenic mammal comprising a cell of claim 28, said mammal showing defective angiogenesis.
 30. An antibody which is specific for an epitope that is specific for a polypeptide of claim
 7. 31. A method of advertising a differentially-regulated human angiogenesis polynucleotide or polypeptide for sale, commercial use, or licensing, comprising, displaying in a computer-readable medium a polynucleotide of claim 1, or a polypeptide encoded thereby. 